One of the primary consequences of ischemia is tissue acidification due to anaerobic production of lactic acid. Upon reperfusion and recovery of pH, cytotoxic edema often ensues. Na+/H+ exchange, a mechanism involved in the regulation of intracellular pH (pHi), is activated by low intracellular pH, is dependent on extracellular Na+, and is inhibited by low extracellular pH (pH less than 6) or by amiloride. In this study we explore the role of Na+/H+ exchange in cell swelling following cytoplasmic acidification of C6 glioma cells. Postischemic intracellular acidification was simulated in vitro by exposure of cells in suspension to: (1) 20 or 140 mM lactic acid; or (2) 10 microM oligomycin. pHi was monitored fluorimetrically using the intracellularly trapped pH-sensitive dye bis(carboxyethyl)carboxyfluorescein. Cell volume was measured electronically with a Coulter Counter/Channelyzer. Both simulations of ischemia caused intracellular acidification followed by recovery. pHi recovery was mediated by Na+/H+ exchange, since it was amiloride-sensitive and Na+-dependent. This pHi reversal following lactic acid-induced acidification was also inhibited at pHo less than 6. Volume measurements showed that cells suspended in 140 mM Na-lactate/lactic acid swelled by 19% over 15 min. This swelling was Na+-dependent, and inhibited by amiloride and pHo less than 6. These results suggest that Na+/H+ exchange may be involved in cell swelling following cytoplasmic acidification, and thus may be involved in postischemic cytotoxic brain edema.