The distribution and chemospecificity of the pedunculonigral neurons have been studied in squirrel monkeys (Saimiri sciureus) with cholera toxin subunit B (CTb) and fluorogold (FG) as retrograde tracers combined with immunohistochemistry for choline acetyltransferase (ChAT), glutamate, and the calcium binding protein calbindin D-28k. The injection of either CTb or FG into the substantia nigra produces prominent retrograde cell labeling in the mesopontine tegmentum. Labeled neurons are particularly numerous at the level of the decussation of the superior cerebellar peduncle, where they abound principally in the pars dissipata of the pedunculopontine nucleus (PPN). A significant proportion of retrogradely labeled neurons in the PPN display ChAT immunoreactivity. Within the entire PPN, approximately 25% of the retrogradely labeled neurons express ChAT immunoreactivity, but proportions of doubly labeled neurons are about 35%, 25%, and 15% in the rostral, middle, and caudal thirds of the PPN, respectively. These doubly labeled neurons are scattered among numerous retrogradely labeled neurons that are ChAT-negative and whose number increases along the rostrocaudal extent of the PPN. Several retrogradely labeled neurons in the PPN display glutamate immunoreactivity, but very few express calbindin. This study provides the first direct evidence for the involvement of cholinergic and glutamatergic neurons in the pedunculonigral projection in primates. Furthermore, the fact that some neurons of the PPN display both ChAT and glutamate immunoreactivity indicates that single neurons in the mesopontine tegmentum may exert a two-fold effect upon dopaminergic neurons of the substantia nigra. This dual cholinergic and glutamatergic pedunculonigral projection may play a crucial role in the functional organization of primate basal ganglia.