Although extensively pursued, the central respiratory neurons have remained elusive. We departed from the more conventional physiologic and morphologic methods of system and tissue examination and cultured dissociated fetal rat cells (Fitzgerald et al., J Neurosci Res 33:579–589, 1992) from the area of the nucleus ambiguus and the nucleus tractus solitarius located within the 2 mm rostral to the obex. Pacemaker-like cells, with a regular single or bursting activity, studied at 3–5 weeks of age, responded to very small pulses of CO2 (50 ms) and low pH with an increase in spike frequency and a decrease in spike amplitude. Other irregularly beating or silent cells did not respond or else required very large pulses (>200 ms) to do so. The pacemaker cells also responded to hypoxia induced by administration of sodium hydrosulfite with an increase in spike frequency and amplitude; high oxygen (>600 torr) and adenosine produced a decrease in electrical activity. Most of these cells were multipolar after staining with antibodies to neuron-specific enolase (NSE) and Fragment C of tetanus toxin. They did not stain for choline acetyltransferase (ChAT). The results suggest that these cultured cells, expressing a phenotype inherently responsive to CO2 and low pH, have the characteristics of central respiratory chemoreceptors, and may be involved in the generation of the respiratory rhythm. © 1992 Wiley-Liss, Inc.