We examined changes in intracellular pH with the fluorescent probe BCECF after treatment with aspirin (ASA) in gastric mucosal cells isolated from guinea pigs at various extracellular, pH levels. The cytotoxic activities of ASA and the intracellular trapping of ASA at various extracellular pH levels were also examined by colorimetric MTT assay and counting the influx of [14C](carboxyl-14C)ASA. Under acid extracellular pH, ASA reduced intracellular pH in gastric mucosal cells and exhibited cytotoxicity. Intracellular trapping of ASA was also observed only under acidic extracellular conditions. The present study suggests that ASA causes gastric mucosal cell injury in vitro by reducing intracellular pH, resulting from intracellular trapping of ASA under acidic extracellular conditions.