The habenular nuclei are the major sources of projections to the interpeduncular nucleus. The habenular neurons of the goldfish are ChAT and NADPH-diaphorase positive. The localization of these two enzymes in the habenulae suggests the involvement of acetylcholine and nitric oxide (the product of NADPH-diaphorase activity in the nervous tissue) in the habenulo-interpeduncular connection. This finding is supported by the presence of ChAT and NADPH-diaphorase activity in the neuropilar area of the interpeduncular nucleus. This activity was depleted 12 days after habenular ablation. The overlap of ChAT and NADPH-diaphorase localization in goldfish habenular neurons is consistent with previous observations on the co-localization of these two enzymes in some brainstem neurons of other vertebrates. It is, however, not identical to the localization of NADPH-diaphorase in the habenulae and in the interpeduncular nucleus of the rat.