In order to clarify the origin and to examine the synaptology of the projection from the mesopontine tegmentum to the entopeduncular nucleus, rats received discrete deposits of anterograde tracers in different regions of the mesopontine tegmentum. Anterogradely labelled fibres in the entopeduncular nucleus were analysed at the light and electron microscopic levels. To determine the neurochemistry of the projection, the distributions of GABA and glutamate immunoreactivity in anterogradely labelled boutons in the entopenducular nucleus were studied by postembedding immunocytochemistry. The morphological characteristics of anterogradely labelled structures were compared to those of choline acetyltransferase-immunopositive structures. The anterograde tracing demonstrated that the projection to the entopeduncular nucleus arises from the area defined by the cholinergic neurons of the pedunculopontine region and from the more medial and largely non-cholinergic, midbrain extrapyramidal area. The anterogradely labelled terminals formed asymmetrical synaptic contacts with dendritic shafts, cell bodies and more rarely spines in the entopeduncular nucleus, and they were significantly enriched in glutamate immunoreactivity compared to identified GABAergic terminals in the same region. The morphology, trajectory and synaptology of the anterogradely labelled fibres showed similarities to those of choline acetyltransferase-immunopositive fibres and terminals, providing indirect evidence in support of previous suggestions that at least part of the projection is cholinergic. The structures postsynaptic to the anterogradely labelled boutons also received input from other classes of terminals that had the morphological and neurochemical characteristics of boutons derived from the neostriatum, globus pallidus and subthalamic nucleus. These findings imply that the mesopontine tegmentum sends a projection to the entopeduncular nucleus that is heterogeneous with respect to its origin and also possibly its neurochemistry. The synaptology of the projection underlies one route through which the mesopontine tegmentum can exert effects on movement by modulating the direct and indirect pathways of information flow through the basal ganglia.