Currently, a change in pHi is believed to be the major signal in the chemosensitive (CS) response of brainstem neurons to hypercapnia; however, multiple factors (e.g., Ca2+, CO2, pHi, and pHo) have been suggested to contribute to this increase in firing rate. While there is evidence for a significant role of pHi in the CS response, we hypothesize that hypercapnic acidosis (HA) can increase firing rate even with no change in pHi. We tested several methods to clamp pHi, including high intracellular buffer and the use of rapid diffusion of weak bases or weak acids through the cell membrane. We were able to clamp pHi during hypercapnic exposure using weak acids. We observed a CS response to HA, with pHi clamped, indicating that intracellular acidification, while sufficient to increase firing rate, is not required for the response of CS neurons. The CS response to HA without a change in pHiis most likely due to extracellular acidification and/or increased CO2 and strongly supports the multiple factors model of chemosensitive signaling.