Substance P stimulates respiration, in part by a direct action on the pre-Bötzinger complex (preBötC). This region of the medulla oblongata contains neurons that are strongly immunoreactive for the neurokinin-1 receptor (NK1R-ir), and a recent theory has postulated that these cells might be the adult form of excitatory interneurons that are essential for respiratory rhythmogenesis in neonates. Here we sought to determine whether preBötC respiratory neurons are indeed NK1R-ir in the adult rat. Preinspiratory (pre-I) neurons were recorded in the preBötC region of halothane-anesthetized rats. Most pre-I cells could be antidromically activated from the contralateral side of the medulla (7 of 10; latency 1.3 ± 0.2 ms), suggesting that most of them were propriomedullary neurons rather than respiratory motoneurons or bulbospinal cells. Thirty-two pre-I neurons including seven cells with contralateral projection were labeled with biotinamide using the juxtacellular method. Eleven cells (34.4%) were NK1R-ir, including three of the seven pre-I cells that were antidromically activated from the contralateral side. In 3 control rats we labeled 20 preBötC neurons with patterns of discharge other than pre-I and found that none were detectably NK1R-ir. In conclusion, some of the intensely NK1R-ir neurons of the adult preBötC region are indeed respiratory interneurons as suggested by Gray et al. The subtype of NK1R identified by the antibody is detectable only in a small minority of preBötC respiratory cells, most notably in pre-I interneurons. Given prior anatomical evidence, these NK1R-ir pre-I interneurons are most likely glutamatergic. The data are consistent with the possibility that the NK1R-ir pre-I interneurons of the adult preBötC could be the adult form of a class of inspiratory neurons that are rhythmogenic in the neonate (either the pacemakers and/or an excitatory subtype of follower neurons).