The expiratory motor neurons of the representative thoracic segment of the rat were examined as to their localization and sites of origin of their premotor neurons in the lower brainstem. Either T6 or T7 was selected as a representative target segment for horseradish peroxidase (HRP) injection. The intercostal motor neurons in the T6 or T7 were doubly labeled by HRP placed in the cut end of the internal intercostal nerve and True blue placed in the cut end of the external intercostal nerve. The thoracic expiratory motor neurons labeled by HRP were concentrated in the oblique zone running along the dorsal to ventrolateral direction in both the T6 and T7 segments. By contrast, the thoracic inspiratory motor neurons labeled by True blue were concentrated in the horizontal zone running along the bottom of the ventral horn in both the T6 and T7 segments. A small amount of HRP was iontophoretically injected through a double-barrel coaxial electrode to sites of the expiratory motor neurons which had been identified electrophysiologically. In 5 successful experiments, the HRP-labeled cells were bilaterally distributed in the para-ambiguus nucleus (59.0%), ventral subnucleus of the paramedian reticular nucleus (13.6%), and raphe nuclei (10.8%). In another experiment, it was found that the expiratory premotor neurons in the para-ambiguus nucleus were present in a narrow column over the entire length of the nucleus at a level between 1.0 mm rostral and 1.4 mm caudal to the obex.