In mammals, the suprachiasmatic nucleus is responsible for the generation of most circadian rhythms and their entrainment to environmental cues. Cholinergic agents can alter circadian rhythm phase, and fibres immunoreactive for choline acetyltransferase, the biosynthetic enzyme for acetylcholine, are present in the suprachiasmatic nucleus. Since there are no cholinergic somata in the suprachiasmatic nucleus, these fibres must represent the terminals of cholinergic neurons whose cell bodies are located elsewhere in the brain. This study was aimed at locating the cholinergic neurons that project to the suprachiasmatic nucleus by retrograde and anterograde tract-tracing and immunohistochemistry for choline acetyltransferase in the rat. After injection of fluorogold, a retrograde tracer, into the suprachiasmatic nucleus, retrogradely labelled neurons that were immunopositive for choline acetyltransferase were located throughout the rostrocaudal extent of the cholinergic basal nuclear complex, with highest densities in the substantia innominata and the nucleus basalis magnocellularis. A few cells were also located in the medial septum and in the vertical and horizontal limbs of the diagonal band of Broca. In the brainstem, double-labelled neurons were located in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus and the parabigeminal nucleus. Injections of the anterograde tracer biocytin in these three brainstem nuclei resulted in fibre labelling in the suprachiasmatic nucleus, consistent with the retrograde findings. No clearly double-labelled cells were located in the retina. These results suggest that the suprachiasmatic nucleus receives cholinergic afferents from both the basal forebrain and mesopontine tegmentum which may mediate cholinergic effects on circadian rhythms.