The ganglion cell layer of the rabbit retina contains neurons that synthesize acetylcholine. To identify these neurons, the ganglion cells were labeled by retrograde transport of a fluorescent dye, and the acetylcholine-synthesizing cells of the same retinas were labeled by exposing the tissue to tritiated choline. Autoradiographs inspected by fluorescence microscopy showed that tritiated acetylcholine and the dye accumulated in different cells. Optic nerves of other animals were sectioned, causing degeneration of many neurons of the ganglion cell layer. This loss affected neither the retina's overall rate of acetylcholine synthesis nor the number of acetylcholine-containing cells in the ganglion cell layer. The acetylcholine-synthesizing neurons thus appear to be displaced amacrine cells.