We employed radioligand binding autoradiography to determine the distributions of pre-and postsynaptic cholinergic radioligand binding sites in the brains of two species of bat, one species of shrew, and the rat. High affinity choline uptake sites were measured with [3H]hemicholinium, and presynaptic cholinergic vesicles were identified with [3H]vesamicol. Muscarinic cholinergic receptors were determined with [3H]scopolamine. The distribution patterns of the three cholinergic markers were simitar in all species examined, and identified known major cholinergic pathways on the basis of enrichments in both pre-and postsynaptic markers. In addition, there was excellent agreement, both within and across species, in the regional distributions of the two presynaptic cholinergic markers. Our results indicate that pharmacological identifiers of cholinergic pathways and synapses, including the cholinergic vesicle transport site, and the organizations of central nervous system cholinergic pathways are phylogenetically conserved among eutherian mammals.