The distribution of cholinergic neurons was studied in painted and red-eared turtles using antisera against choline acetyltransferase (ChAT). The results showed that the organization of cholinergic systems in the central nervous system of turtles was remarkably similar to that previously described in diverse other species. For example, the present studies revealed the presence of cholinergic neurons in the striatum, the basal telencephalon, the isthmo-tectal system, and a region of cells near the cerebellar peduncle. The motoneurons of the III, IV, V, VI, VII, X and XII cranial nerves and the spinal ventral horn were also shown to be cholinergic. Additional cholinergic neurons were observed in the hypothalamus and rhombencephalic reticular formation. These results help to clarify the precise extent and location of the major cholinergic cell groups of the turtle brain, thereby adding to our understanding of the organization of the turtle nervous system. The results also reveal that the cholinergic systems of the striatum, basal forebrain, isthmic region, and cerebellar peduncular region are ancestral features of the amniote brain and are common to all living amniotes that have been studied. Thus, the organization and, presumably, the function of the cholinergic systems appear to be conservative.