BIOELECTRIC activity of CO2-sensitive, ventrolateral medullary neurons (VLNCS) in organotypic cultures from the obex level of newborn rats was tested during changes of the intracellular pH (pHi) measured in BCECF-AM loaded cultures. Hypercapnia (pCO280–100 mmHg) reduced pHiby 0.15 ± 0.06 units and stimulated neuronal discharges. Replacement of CO2/HCO3−in the bath by HEPES (26 mM, pH 7.4) for 10 min acidified pHi(0.07 ± 0.03 units) and also excited VLNCS. Ammonium chloride (10 mM, 1 min) initially alkalized (0.1 ± 0.04) and thereafter acidified pHi(0.06 ± 0.03), while the extracellular pH was first acidified and then alkalized. This resulted in neuronal discharge which were first suppressed and then accelerated. The findings strongly suggest that intracellular rather than extra-cellular acidification activates CO2-sensitive neurons.