In all vertebrates, including humans, the Edinger-Westphal nucleus (EW) forms a circumscribed cell group dorsomedial to the oculomotor nucleus (nIII). Traditionally the EW is considered the location of parasympathetic preganglionic neurons of the ciliary ganglion, mediating pupillary constriction and accommodation. In a comparative study in rat, ferret, monkey, and human, the location of cholinergic neurons within and around the nIII, which includes motoneurons of the extra-ocular muscles and the preganglionic neurons of the ciliary ganglion, was compared to the location of urocortin-positive neurons. Irrespective of the species, the cholinergic and urocortin-positive neurons form largely separated cell populations adjacent to each other. Only in monkey, cholinergic putative preganglionic neurons were found within the cytoarchitecturally defined EW, whereas in rat, ferret, and human the EW is almost exclusively composed of urocortin-positive neurons. In humans, the presumed preganglionic neurons are located as an inconspicuous group of choline acetyltransferase-positive neurons dorsal to the urocortin-positive EW.