A 23-kDa, soluble, calcium-binding photoreceptor-specific protein (23-kDa) has been shown to be identical to recoverin and the cancer-associated retinopathy protein. Recoverin has been reported to activate guanylate cyclase to increase the amount of cyclic GMP and thereby reopen cation channels within the photoreceptor cells. In this study, the 23-kDa protein was purified from bovine retinas and monospecific antibodies against it were generated in rabbits. Western analysis demonstrated 23-kDa in retinas from human, monkey, bovine, dog, rabbit, rat, mouse, frog, chameleon and iguana although it was not detected in chicken or fly retinas. No immunoreactivity was observed in any non-retinal tissues except the pineal gland. The 23-kDa protein was detected, by Western analysis, at postnatal day 5 in the mouse retina and it increased in amount in parallel with the differentiation of the photoreceptor cells in normal mice and it also decreased in parallel with their degeneration in the rd mouse. Immunocytochemical analysis of the adult mouse retina showed that 23-kDa is restricted primarily to the inner segments of the photoreceptor cells and, unlike arrestin, its localization did not shift in response to light/dark changes.