The birefringence (deltan) of Rana pipiens rod outer segments (ROS) reveals microstructure inhomogeneities not seen with other techniques. In the basal 20-30-micron length of the ROS there is a nearly linear axial gradient in deltan of approximately equal to -2 x 10(-5)/micron. No consistent deltan gradients are found in the distal 20-30 micron of the ROS. Using glycerol imbibition to separate the intrinsic and form birefringence components, we found that the basal deltan gradient was principally due to a gradient of the intrinsic birefringence component. The disk membrane volume fraction decreases uniformly from the basal end to the distal end, while the disk membrane refractive index increases. The contributions of these changes to the form birefringence approximately cancel, so that the form component is fairly uniform along the ROS axis. Because its axial distance from the inner segment is a measure of the time since a disk membrane was formed, these gradients may reflect a disk membrane aging process. Occasionally a highly birefringent, 2-micron-wide band is seen at the basal end ot the ROS, possibly where the envelope membrane folds to form new disk membranes.