Background: An assay of the enzyme activity is very important for diagnosis of metabolic disorder. The activity of methylmalonyl-CoA mutase has been measured using fibroblast. However, the collections of the fibroblast from the patient and cell culture are troublesome task. We examined the simple and easy method using lymphocyte. Method: A mixture of methylmalonyl-CoA, lymphocyte and cobalamin in Tris-sulfate buffer (pH 7.5) was incubated for 15 min at 37°C. After the suspension was centrifuged (12,000 rpm, 10 min), the supernatant was analyzed by HPLC. STR-ODS 2 (6×150 mm, Shinwakagaku, Japan) was used as the HPLC column and detection of methylmalonyl-CoA and succinyl-CoA was performed by UV absorption (260 nm). Results: Although succinyl-CoA was detected in lymphocytes from a healthy volunteer, none or a small amount of succinyl-CoA was generated in the lymphocytes of a patient with methylmalonic acidemia. The amount of generated succinyl-CoA was proportinal to methylmalonyl-CoA mutase activity. Discussion: The measurement of methylmalonyl-CoA mutase activity with lymphocyte was possible. However, as succinyl-CoA decomposed with time by heat, analysis has to be performed carefully.