We have studied the distribution of the calcium-binding protein calbindin in the adult rabbit retina by using a commercially available antibody and immunocytochemical methods. The most heavily labeled cells are A-type horizontal cells, but B-type horizontal cells are also lightly labeled by this antibody. Among the horizontal cells, there is a mosaic of small, well-labeled somata, which we have identified as a subset of ON cone bipolar cells. In addition, some wide-field amacrine cells and a few large ganglion cells are also labeled for calbindin. The calbindin bipolar cells form a regular mosaic with a peak density of approximately 1,700 cells/mm2, falling to 550 cells/mm2 in the periphery. They account for about one-twelfth of cone bipolar cells, and they are narrowly stratified deep in sublamina 4 of the inner plexiform layer immediately above the rod bipolar terminals. Double-label experiments using an antibody to protein kinase C (PKC) indicate that the calbindin bipolar cells are completely distinct from the population of rod bipolar cells. Rod bipolar cells outnumber the calbindin cone bipolar cells by a factor of four to five. Further double-label experiments show that the calbindin bipolar cells are also labeled for recoverin. The calbindin bipolar cells are well coupled to AII amacrine cells, and they account for roughly 23% of the AII coupled bipolar cells. This suggests that there are three to four additional ON cone bipolar cell types that are coupled to AII amacrine cells. The calbindin cone bipolar cell described in this paper shares many characteristics with a reconstructed cone bipolar cell that forms the most gap junctions with AII amacrine cells (Strettoi et al. [1994] J. Comp. Neurol. 347:139-149). We conclude that these different methodologies provide complementary descriptions of the same cone bipolar cell type. The calbindin antibody defines a subset of cone bipolar cells in the rabbit retina. The cells in this subset are almost certainly the deepest of the cone bipolar cells. The tight stratification of the calbindin cone bipolar cell suggests that the inner plexiform layer is stratified according to depth, with narrow functional divisions within the broad partition of sublamina b, where ON signals are processed. The strength of coupling between the calbindin cone bipolar cells and AII amacrine cells suggests this pathway plays a major role under scotopic conditions.