We determined the regional intracellular pH (pHi) of rat brainstem in response to hypercapnia. Neutral red spectrophotometry was used to characterize changes in intracellular pH along the dorsal and ventral aspects of the medulla oblongata. Male Wistar rats (250-350 g) were infused with 3 ml of 2% Neutral Red over the course of 30 minutes. After 20 minutes of infusion, the rats were ventilated with either 7% CO2, 21% O2 in N2 or room air for 15 minutes. Our data indicate that hypercapnia induces prominent intracellular acidification in neurons within putative chemosensitive regions of the ventral aspect of medulla oblongata. On the contrary, minimal or no changes were observed in neurons within the nucleus tractus solitarius. These findings suggest that brainstem neurons differentially regulate intracellular pH during hypercapnic stress.