We investigated the morphology, distribution and synaptic connectivity of cholinergic neurons in the mouse retina by immunocytochemistry, using antisera against choline acetyltransferase (ChAT). ChAT-immunoreactive amacrine cells fall into two groups according to the localization of their somas in the retina: one is situated in the inner nuclear layer (INL), near the border of the inner plexiform layer (IPL), and the other is displaced in the ganglion cell layer (GCL). The dendrites of amacrine cells from the INL ramify in sublamina a and that of the displaced amacrine cells ramify in sublamina b of the IPL. Double labeling with an antisera against ChAT and r-aminobutyric acid (GABA) demonstrated that these labeled cells formed a subpopulation of GABAergic amacrine cells. The synaptic connectivity of ChAT-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The most frequent synaptic input of ChAT-labeled amacrine cells was from bipolar cells in both sublaminae a and b of the IPL, followed by labeled amacrine cells and unlabeled amacrine cells. Their primary output targets were onto ganglion cells in both sublaminae a and b and output onto ganglion cells was more frequently observed in sublamina b of the IPL. Our results suggest that cholinergic amacrine cells in the mouse retina are very similar to their counter parts in other mammals, and they can attribute a major role in the pathway feeding into directionally selective ganglion cells.