Further evidence that substance P (SP) is a neurotransmitter of baro- and/or chemoreceptor afferent nerves has been obtained by 1) measuring the (SP) content of the nucleus tractus solitarius (NTS) in the rat following unilateral nodose ganglionectomy 2) utilizing immunocytochemistry to detect the presence of SP in peripheral baro- and chemoreceptive regions and pathways of the afferent fibers and 3) investigating the potassium stimulated release of SP from the NTS in vitro. It was found that the NTS contained significant amounts of SP-I and that unilateral removal of the nodose ganglia reduces the SP-I content of those portions of the NTS known to receive vagal afferents. In addition, SP-I was visualized in discrete fibers in the tunica adventitia of the aortic arch and carotid sinus regions, the carotid body, the vagus nerve and nodose ganglia. Stimulation of freshly dissected slices of the NTS region with 50 mM potassium in the presence of calcium increases SP release 2-3 fold. Omission of calcium from the buffer completely abolished SP release by K ion. These results in the rat are consistent with our previous studies in the cat and provide further evidence that SP is contained within baro- and chemoreceptor afferent nerves.