Cholinergic neurons in the retina of the tree shrew were identified immunocytochemically using a monoclonal antibody directed against choline acetyltransferase (ChAT). The chief result is that roughly 4 times as many ChAT-immunoreactive neurons are found in the inner nuclear layer (INL) as in the ganglion cell layer (GCL). In the INL, two classes of cholinergic neuron can be distinguished on the basis of soma size, one large and one small. The large neurons correspond closely in size and number to the displaced cholinergic neurons in the GCL, suggesting that these are the matching populations of cholinergic amacrine cells reported in other species. The small ChAT-immunoreactive neurons, on the other hand, which make up 60% of the total number of ChAT-positive neurons in the retina, appear to have no counterpart in the GCL. Whether these small neurons are a separate class of amacrine cell or some other cell type (e.g. bipolar, interplexiform, etc.) remains to be determined.