Cholinergic amacrine cells in the tiger salamander retina were observed for the first time by using antibodies against choline acetyltransferase (ChAT). ChAT-immunoreactive cells were present in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), and the somas of the former population (average diameter = 15.13 μm) were slightly smaller than those of the latter population (average diameter = 16.42 μm). The processes of these cells form two distinct narrow bands in the inner plexiform layer (IPL), one located near 0.2 inner plexiform units (IU) and the other near 0.65–0.7 IU. Soma size, cell density and spatial distribution of ChAT-positive cells were quantitatively analyzed. Our results suggest that cholinergic amacrine cells in the salamander retina are very similar to their counter parts in other species, and they can be used as a model system for studying cholinergic functions in the visual system.