Golgi studies of cat retina have revealed the presence of matching subpopulations of starburst-like amacrine and displaced amacrine cells that are morphologically similar to the cholinergic cells of rabbit retina. The displaced amacrines appear identical with the A14 cells described by Kolb et al. (Kolb, Nelson, and Mariani: Vision Res. 21:1081-1114, 1981). In order to determine whether these cells may be cholinergic, we carried out autoradiography to localize newly synthesized (3H)acetylcholine and immunocytochemistry to demonstrate the distribution of choline acetyltransferase. Autoradiographs showed labeling in somas of both amacrine and displaced amacrine cells. Choline acetyltransferase was found in amacrine cells that ramify in sublamina a of the inner plexiform layer and in displaced amacrine cells ramifying in sublamina b. The pattern of cholinergic neurons in the cat is similar to that in other vertebrates and suggests that acetylcholine may play an important and consistent role in retinal function.