Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnia. Currently, pHi is believed to be the major signal in the chemosensitive (CS) response; however, a new model has been proposed suggesting that multiple factors (such as Ca++, CO2, pHi, and pHo) contribute to the CS response. While there is evidence for a significant role of pHi in the CS response, we hypothesize that hypercapnic acidosis (HA) can still increase FR even with no change in pHi. To test this hypothesis, we developed a method to clamp pHi using rapid diffusion of the weak acid, acetic acid, through the cell membrane according to the relationship: pHi = pHo + log ([C2H3O2] i/[C2H3O2]o). Pontine brainstem slices (300 µm) were cut from neonatal rat pups (P4-P16). FR of LC neurons was measured with whole cell patch pipettes, pHi was measured by fluorescence imaging using the dye pyranine, and neurons were loaded from the patch pipette with 50 mM acetate. Extracellular acetate was varied to set pHiat desired values using the weak acid diffusion technique. We eliminated pHidecreases (0.07 ± 0.06 pH unit, p>0.05, n=8) of LC neurons upon exposure to HA (15% CO2, pHo 7.0) compared to the normal response (0.25 ± 0.03 pH unit, pi clamping, HA induced an increase in FR (CS index = 144 ± 14%, n=7) similar to controls (140 ± 5%, n=17). While a decrease in pHi is sufficient to elicit the chemosensitive response, these data indicate that intracellular acidification is not required. A chemosensitive response to hypercapnia without a change in pHi strongly supports the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].