The distribution of cholinergic neurons in the region of the cat Edinger-Westphal nucleus (EW) was determined by immunocytochemical localization of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT). Neurons containing ChAT-like immunoreactivity (ChAT-LI) were densely distributed within EW, the anteromedian nucleus (AM), and the oculomotor nucleus (III), and were also present in immediately adjacent regions of the periaqueductal gray and ventral tegmental region. The majority of labelled neurons in EW and AM showed a markedly lower intensity of ChAT-LI than the labelled neurons in III and adjacent regions. To determine the relationship of cells with ChAT-LI to the distribution of ciliary ganglion afferent neurons, a double labelling immunocytochemistry/retrograde transport technique was also used. These experiments showed that many of the cells located outside of III that stained intensely for ChAT-LI project to ciliary ganglion. Very few ciliary ganglion afferent neurons were found in EW or AM itself; instead, the distribution of lightly labelled ChAT-LI-positive neurons in EW and AM more closely matched the known distribution of peptide-containing cells that have descending, central projections.