A focal position determining method for determining a focal position of an objective lens (30, 30') focused on an observed target region (10a, 10'a) in a specimen (10, 10') including a living cell that emits a feeble light, comprising: a light irradiating step (SA-1) of irradiating light to the specimen (10, 10'); a focal position changing step (SA-2) of changing the focal position of the objective lens (30, 30'); a focal position measuring step (SA-3) of measuring the focal position that is changed at the focal position changing step (SA-2); a specimen imaging step (SA-4) of imaging the specimen (10, 10') to which light is irradiated at the light irradiating step (SA-1), at the focal position changed at the focal position changing step (SA-2); a feature data calculating step (SA-5) of calculating feature data characterizing an imaged image based on an image taken in transmission at the specimen imaging step (SA-4), wherein the feature data is indicative of a contrast value of the imaged image; an executing step (SA-6) of repeatedly executing the focal position changing step (SA-2), focal position measuring step (SA-3), specimen imaging step (SA-4), and feature data calculating step (SA-5); a focal position selecting step (SA-7) of selecting at least one focal position from the plural focal positions accumulated by the execution of the executing step (SA-6) based on the plural feature data accumulated by the execution; and a focal position determining step (SA-8) of determining the focal position of the objective lens (30, 30') focused on the observed target region (10a, 10'a) based on the focal position selected at the focal position selecting step (SA-7), characterized in that at the focal position selecting step (SA-7), two focal positions are selected from the plural focal positions accumulated by the execution of the executing step (SA-6) based on the plural feature data accumulated by the execution, wherein the selected two focal positions are focal positions of the objective lens (30, 30') having the feature data corresponding to two local maximum contrast values, and at the focal position determining step (SA-8), the central position between the two focal positions is determined as the focal position focused on the observed target region (10a, 10'a) based on the two focal positions selected at the focal position selecting step (SA-7). a light irradiating step (SA-1) of irradiating light to the specimen (10, 10'); a focal position changing step (SA-2) of changing the focal position of the objective lens (30, 30'); a focal position measuring step (SA-3) of measuring the focal position that is changed at the focal position changing step (SA-2); a specimen imaging step (SA-4) of imaging the specimen (10, 10') to which light is irradiated at the light irradiating step (SA-1), at the focal position changed at the focal position changing step (SA-2); a feature data calculating step (SA-5) of calculating feature data characterizing an imaged image based on an image taken in transmission at the specimen imaging step (SA-4), wherein the feature data is indicative of a contrast value of the imaged image; an executing step (SA-6) of repeatedly executing the focal position changing step (SA-2), focal position measuring step (SA-3), specimen imaging step (SA-4), and feature data calculating step (SA-5); a focal position selecting step (SA-7) of selecting at least one focal position from the plural focal positions accumulated by the execution of the executing step (SA-6) based on the plural feature data accumulated by the execution; and a focal position determining step (SA-8) of determining the focal position of the objective lens (30, 30') focused on the observed target region (10a, 10'a) based on the focal position selected at the focal position selecting step (SA-7), characterized in that at the focal position selecting step (SA-7), two focal positions are selected from the plural focal positions accumulated by the execution of the executing step (SA-6) based on the plural feature data accumulated by the execution, wherein the selected two focal positions are focal positions of the objective lens (30, 30') having the feature data corresponding to two local maximum contrast values, and at the focal position determining step (SA-8), the central position between the two focal positions is determined as the focal position focused on the observed target region (10a, 10'a) based on the two focal positions selected at the focal position selecting step (SA-7).