Using a monoclonal antibody against choline acetyltransferase, neurons of the rat entopeduncular nucleus were found to express choline acetyltransferase immunoreactivity. These cholinergic cells were located mostly in the rostral portion of the entopeduncular nucleus with a marked decrease towards its caudal portion. To identify their target sites, a retrograde fiber tracing technique was combined with immunohistochemistry for choline acetyltransferase. After injection of wheatgerm agglutinin conjugated with horseradish peroxidase into the habenula, some of the entopedunculo-habenular cells were found to be immunoreactive for choline acetyltransferase. The cells in the peripallidal region (the substantia innominata, nucleus basalis magnocellularis and ansa lenticularis) with choline acetyltransferase immunoreactivity did not contain horseradish peroxidase. Following injection of fluorescent tracer into the frontal cerebral cortex, retrogradely labeled cells were observed in the rostral part of the entopeduncular nucleus. A majority of these entopedunculo-cortical cells exhibited choline acetyltransferase immunoreactivity, similar to the cells of the peripallidal region projecting to the neocortex. Employing two different fluorescent tracers, entopedunculo-cortical cells were shown to constitute a distinct cell population from the numerous entopedunculo-habenular cells. The present study demonstrated, in the rat entopeduncular nucleus, the presence of cholinergic neurons that projected to the neocortex and habenula.