On the basis of previous studies demonstrating a wide colocalization of NADPH-diaphorase (ND) and choline acetyltransferase (ChAT) in mammalian and reptilian brainstem, ND histochemistry and ChAT immunocytochemistry have been combined to study the distribution of both markers in the mesopontine region of an avian species, the Japanese quail (Coturnix japonica). Go-existence of the two neurochemical markers is present only in part of the system, namely, in the nucleus tegmentalis pedunculo-pontinus, in the nucleus tegmentalis latero-dorsalis, in the nucleus mesencephalicus profundus, and in the nucleus reticularis paragigantocellularis. The degree of colocalization varies in these regions from about 50 to 95% of the ChAT population. However, several other nuclei in the same region display only one of the two markers. These results confirm that even if the general distribution of the ND-positive neurons is largely comparable in vertebrates, there exists species-specific differences in the extension of the system and in the degree of colocalization with other neurochemical markers. (C) 1997 Elsevier Science Ireland Ltd.