A combined fluorescent retrograde tracing and acetylcholinesterase (AChE) histochemical technique was used for the study of some forebrain projections to the interpeduncular nucleus (IPN). After injections of a fluorescent tracer into the IPN, the distribution of AChE-containing and of fluorescent retrogradely labeled neurons was simultaneously studied in the habenular nuclei, medial septum and diagonal band of Broca. In all these regions, the majority of retrogradely labeled neurons also contained AChE: neurons located in the habenular nuclei stained lightly or moderately for the enzyme, while neurons located in the diagonal band and medial septum displayed intense AChE staining and were classified as putatively cholinergic perikarya. In all regions, a minority of labeled neurons did not stain for AChE, and were identified as non-cholinergic neurons projecting to the IPN. The present study shows the existence of a biochemical heterogeneity in the habenulo-interpeduncular and telencephalo-interpeduncular pathways, and indicates that the latter contains putatively cholinergic as well as non-cholinergic fibers.