Electrolytic lesions and surgical transection of the habenulo-interpeduncular-ventrotegmental tract have established the existence of separate habenulo-interpeduncular-ventrotegmental substance P and cholinergic projections. Micro-knife lesions separating the habenula nuclei showed the medial habenular nucleus to be the source of substance P fibres running via the fasciculus retroflexus to the ventral tegmental area. The lateral habenular nucleus receives a substance P projection from the medial habenular nucleus and is the source of cholinergic projection to the interpeduncular nucleus and to the medial habenular nucleus. Lesions of the ventrotegmental-interpeduncular area did not modify the levels of substance P and choline acetyltransferase in the habenula. These observations suggested that there are no substance P or ACh containing afferents to the habenula from the ventrotegmental-interpeduncular area and the accumulation of substance P and AChE proximal to but not caudal to transections of the fasciculus retroflexus confirmed this view.