Several lines of evidence suggest that the cholinergic neurons of the mesopontine tegmentum contain elevated levels of glutamate and are the source of cholinergic terminals in the subthalamic nucleus and entopeduncular nucleus. The object of this study was to test whether cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, also express relatively high levels of glutamate. To address this, double immunocytochemistry was performed at the electron microscopic level. Perfuse-fixed sections of rat brain were immunolabelled to reveal choline acetyltransferase by the pre-embedding avidin-biotin-peroxidase method. Serial ultrathin sections of cholinergic terminals in both the entoped uncular nucleus and subthalamic nucleus were then subjected to post-embedding immunocytochemistry to reveal glutamate and GABA. Quantification of the immunogold labelling showed that choline acetyltransferase-immunopositive terminals and boutons in both regions were significantly enriched in glutamate immunoreactivity and had significantly lower levels of GABA immunoreactivity in comparison to identified GABAergic terminals. Furthermore, the presumed transmitter pool of glutamate i.e. that associated with synaptic vesicles, was significantly greater in the choline acetyltransferase-positive terminals than identified GABA terminals, albeit significantly lower than in established glutamatergic terminals. In the entopeduncular nucleus, a small proportion of cholinergic terminals displayed high levels of GABA immunoreactivity. Taken together with other immunocytochemical and tracing data, the elevated levels of glutamate in cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, is further evidence adding weight to the suggestion that acetylcholine and glutamate may be co-localized in both the perikarya and terminals of at least a proportion of neurons of the mesopontine tegmentum.