Ascending projections from the pedunculopontine tegmental nucleus (PPT) and the surrounding mesopontine tegmentum to the forebrain in the rat are here examined by using both retrograde and anterograde tracing techniques combined with choline acetyltransferase (ChAT) immunohistochemistry. The anterogradely transported lectin Phaseolus vulgaris-leukoagglutinin (PHA-L) was iontophoretically injected into the PPT in 12 rats. Anterogradely labelled fibers and varicosities were observed in the thalamic nuclei, confirming the findings of our previous retrograde studies (Hallanger et al: J. Comp. Neurol. 262:105-124, '87). In addition, PHA-L-labelled fibers and varicosities suggestive of terminal fields were observed in the anterior, tuberal, and posterior lateral hypothalamic regions, the ventral pallidum in the region of the nucleus basalis of Meynert, the dorsal and intermediate lateral septal nuclei, and in the central and medial nuclei of the amygdala. To determine whether these were cholinergic projections, the retrograde tracer WGA-HRP was injected into terminal fields in the hypothalamus, septum, ventral pallidum, and amygdala. Numerous ChAT-immunoreactive neurons in the PPT and laterodorsal tegmental nucleus (LDT) were retrogradely labelled from the lateral hypothalamus. These cholinergic neurons constituted over 20% of those retrogradely labelled in the dorsolateral mesopontine tegmentum; the balance consisted of noncholinergic neurons of the central tegmental field, retrorubral field, and cuneiform nucleus. Following placement of WGA-HRP into dorsal and intermediate lateral septal regions, the vast majority (greater than 90%) of retrogradely labelled neurons were cholinergic neurons of the PPT and LDT, with few noncholinergic retrogradely labelled neurons in the adjacent tegmentum. In contrast, fewer cholinergic neurons were retrogradely labelled following placement of tracer into the nucleus basalis of Meynert or into the central, medial, and basolateral nuclei of the amygdala, while numerous noncholinergic neurons of the central tegmental field rostral to the PPT and of the retrorubral field adjacent to the PPT were retrogradely labelled in these cases. These anterograde and retrograde studies demonstrate that cholinergic PPT and LDT neurons provide a substantial proportion of mesopontine tegmental afferents to the hypothalamus and lateral septum, while projections to the nucleus basalis and the amygdala are minimal.