This study demonstrates the pedunculopontine tegmental nucleus (PPN) to be the source of a major cholinergic projection to the rat substantia nigra (SN). Neurons of the PPN were double-labeled utilizing choline acetyltransferase immunocytochemistry combined with retrograde transport of horseradish peroxidase (HRP). The cholinergic projection to the SN originates from neurons located in predominately the ipsilateral and rostral portions of the PPN. Other cholinergic neurons that were also retrogradely labeled with HRP were located in the caudal PPN and in the lateral dorsal tegmental nucleus. A non-cholinergic projection from this region was also identified. These findings indicate that the PPN may be topographically organized with respect to its efferent projections. In addition, this study provides evidence for an extrinsic source of acetylcholine to the basal ganglia and implicates the PPN as a source of potentially significant influence over basal ganglia function.