Staining of rat retinal wholemounts with a monoclonal antibody against choline-acetyl-transferase (ChAT) reveals two matching populations of amacrine cells in pigmented and albino rat retinae. One population has cell bodies in the inner nuclear layer (INL). Their dendrites are confined to a narrow stratum in the outer half of the inner plexiform layer (IPL). The other, displaced, population has cell bodies in the ganglion cell layer (GCL) with dendrites stratifying in the middle of the IPL. The density changes with eccentricity, ranging from 1,700 cells/mm2 centrally to 600 cells/mm2 in the periphery. Presumptive cholinergic cells were filled with the fluorescent dye Lucifer yellow. Both subpopulations have the same "starburstlike" morphology as described for rabbit cholinergic amacrine cells (Famiglietti, '83; Tauchi and Masland, '84; Masland et al., '84b). Their dendritic tree sizes change with eccentricity and range from 160 to 300 microns in diameter. Counterstaining of Lucifer yellow-filled cells by ChAT immunohistochemistry did not yield an unequivocal double staining. Nevertheless, indirect evidence of same soma size, same number and form of primary dendrites, same level of stratification, and the good fit into the cholinergic mosaic makes it very likely that the "starburstlike" amacrine cells in the rat use acetylcholine as their transmitter. A comparison with the rabbit cholinergic system strengthens this assumption and reveals a striking similarity between both species.