The subthalamic nucleus (STN) receives cholinergic and non-cholinergic projections from the mesopontine tegmentum. This study investigated the numbers and distributions of neurons involved in these projections in rats using Fluorogold retrograde tracing combined with immunostaining of choline acetyltransferase and a neuron-specific nuclear protein. The results suggest that a small population of cholinergic neurons mainly in the caudoventral part of the pedunculopontine tegmental nucleus (PPN), approximately 360 neurons (≈10% of the total) in the homolateral and 80 neurons (≈2%) in the contralateral PPN, projects to the STN. In contrast, the number of non-cholinergic neurons projecting to the STN was estimated to be nine times as much, with approximately 3300 in the homolateral side and 1300 in the contralateral side. A large gathering of the Fluorogold-labeled non-cholinergic neurons was found rostrodorsomedial to the caudolateral PPN. The biotinylated dextran amine (BDA) anterograde tracing method was used to substantiate the mesopontine–STN projections. Injection of BDA into the caudoventral PPN labeled numerous thin fibers with small en-passant varicosities in the STN. Injection of BDA into the non-cholinergic neuron-rich area labeled a moderate number of thicker fibers with patches of aggregates of larger boutons. The densities of labeled fibers and the number of retrogradely labeled cells in the mesopontine tegmentum suggested that the terminal field formed in the STN by each cholinergic neuron is more extensive than that formed by each non-cholinergic neuron. The findings suggest that cholinergic and non-cholinergic mesopontine afferents may carry different information to the STN.