To evaluate endocytosis in rod photoreceptor inner segments, isolated retinas from Xenopus laevis were maintained in vitro in the presence of the tracer, horseradish peroxidase (HRP). HRP-positive vesicles, 110 +/- 82 nm in diam. (mean +/- S.D.), appeared in the inner segment cytoplasm within 2 min of incubation. Within 8-15 min, 20-30% of the vesicles in the ellipsoid were HRP-positive, reaching approx. 50% by 30-60 min and maintaining this level through 4 hr of culture. In retinas incubated 30 min with HRP and then cultured for an additional period without tracer, the HRP-labeled vesicles were reduced in number by 50- and 75% in the 1- and 2-hr chase incubations, respectively. In retinas cultured for 8 min or longer, multivesicular bodies in the inner segments also contained HRP reaction product, suggesting that the HRP-labeled endocytotic vesicles enter multivesicular bodies. Retinas cultured with HRP for greater than or equal to 1 hr contained significantly more vesicles (per unit area of ellipsoid cytoplasm) as compared with unincubated retinas, retinas incubated from 1 hr in normal media or retinas incubated 30 min with HRP followed by normal chase medium for greater than or equal to 30 min. Taken together, these data indicate that the rod photoreceptor inner segment is capable of extensive endocytotic activity, a process which may function to recover components of the inner segment plasma membrane and the interphotoreceptor matrix.