Abstract Three technically independent approaches have been used to identify cholinergic synapses of the rodent's habenulo-interpeduncular tract. These involved electron-microscopic examination of the interpeduncular nucleus to determine (i) the numbers of degenerating vs normal boutons 2 to 3 days after placement of electrolytic lesions in the habenula; (ii) the distribution of silver grains 1 day after stereotaxic injections of [ 3H]leucine into the medial habenula; and (iii) the type of structures stained for choline acetyltransferase using a peroxidase-antiperoxidase immunohistochemical technique. There is only one type of bouton in the interpeduncular nucleus which degenerated after such lesions in the habenula, was preferentially labeled by axonally transported 3H-labeled protein from the medial habenula, and was specifically stained for choline acetyltransferase. This type contained round vesicles (40 to 60 nm in diameter) moderately accumulated near the presynaptic membrane, and made single or multiple asymmetrical contacts with major dendrites. They often made a paired synapse on a common thin dendritic protrusion. These boutons were morphologically more like the hippocampal boutons, which have been ascribed to long-axoned septohippocampal cholinergic neurons, than they were like the striatal interneuronal cholinergic synapses.