Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte balance. Immunohistochemical studies have shown that large, multipolar atriopeptin-like immunoreactive (APir) neurons are present in areas of the midbrain corresponding to the large neurons of the pedunculopontine tegmental (PPT) and lateral dorsal tegmental (TLD) nuclei, all of which can be stained immunohistochemically for choline acetyltransferase-like immunoreactivity (ChATir). A subpopulation of these cholinergic PPT and TLD neurons are also known to contain substance P-like immunoreactivity (SPir). Using an immunofluorescent technique that allows simultaneous localization of two antigens, we have studied the relationship between APir, SPir and ChATir in the pontine tegmentum of the rat. We have found that the large multipolar APir neurons of the pontine tegmentum are identical to the ChATir neurons of the PT and TLD nuclei and a subpopulation of the APir neurons in PPT and TLD neurons are also SPir.