The birefringence of frog retinal rod outer segments is analyzed in terms of a three-dielectric layer model. The possibility that the birefringence gradient found in such cells is due to changes in the disk membrane-pair spacing is investigated using previously published glycerol imbibition data (Kaplan et al., 1978. Biophys. J. 23: 59-70). The higher net birefringence of the basal end compared to the midpoint of rod outer segments can be accounted for by a smaller negative form birefringence term due to either a smaller or larger intradiskal space, depending upon the assumed relative solids contents of the intradiskal and cytoplasmic spaces.