Choline acetyltransferase (ChAT)-like immunoreactivity in the human retina can be demonstrated using a polyclonal antiserum to ChAT isolated from chick brain. There is a population of ChAT-like immunoreactive cells along both margins of the inner plexiform layer (IPL). The labeled cells have a morphology and position characteristic of the cholinergic amacrine- and displaced amacrine cells demonstrated by other workers in the mammalian retina. Non-immune rabbit serum or pre-absorbed antiserum, used in place of the primary antiserum, verified the specificity of the method. Human retinas can also be labeled with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI), which has been reported to bind selectively to DNA in the nuclei of cholinergic cells. The fluorescent cells are similar in morphology, position, and distribution to the cells which show ChAT-like immunoreactivity. In addition, we have localized the presence of [3H]choline and [3H]choline metabolites in freeze-dried, vapor-fixed tissue using 'dry' autoradiographic techniques. Incubation in [3H]choline was followed by either stimulation or inhibition of calcium-dependent transmitter release during a 1-hr 'chase' period. Using tissue incubated in a chase designed to retain labeled neurotransmitters, silver grains were concentrated over a population of cell bodies at either margin of the IPL (i.e. in the same position as putative ChAT-immunoreactive cells and DAPI-labeled cells). In contrast, tissue incubated in a chase designed to release labeled acetylcholine was labeled uniformly throughout the neural retina, with a heavy band of label over the pigment epithelium. Taken together, the results presented here indicate that three independent markers for cholinergic cells (i.e. ChAT immunoreactivity, DAPI binding, and choline uptake) are present in a population of cells in the human retina. This suggests that acetylcholine may be a neurotransmitter synthesized by amacrine and displaced amacrine cells in the retina.