OBJECTIVE Acidity of brain intracellular and extracellular fluids appears to increase brain injury from stroke, but low extracellular pH decreases the activity of N-methyl-D-aspartate receptor ion channels and decreases calcium influx into isolated neurons. To further investigate the role of acid-base balance in hypoxic brain injury, we studied the influences of intracellular and extracellular pH on calcium influx in cortical brain slices during hypoxia. METHODS Intracellular calcium ([Ca2+]i) and pH (pHi) were measured fluorometrically with the dyes fura-2 and biscarboxyethyl carboxyfluorescein, respectively, during two types of hypoxia: (1) slice perfusate equilibrated with N2/CO2 at pH 6.6 or 6.2 ("gaseous hypoxia") or (2) perfusate equilibrated with 95% O2/5% CO2 plus 100 mumol/L NaCN at pH 7.3, 6.6, or 6.2 ("chemical hypoxia"). RESULTS Changes in perfusate pH under aerobic conditions did not change [Ca2+]i. However, influx of calcium caused by gaseous or chemical hypoxia increased significantly with decreasing perfusate pH. During chemical hypoxia, the elevation in [Ca2+]i at perfusate pH 6.2 was twice that at perfusate pH 7.3. Change in [Ca2+]i was correlated with perfusate pH but not pHi. CONCLUSIONS These results, which differ from previous studies showing acid inhibition of calcium influx in isolated neurons, suggest that low extracellular pH may exacerbate cellular injury during severe hypoxia or ischemia in the intact brain.