The effects of acetylsalicylic acid on gastric mucosal ATP and phosphocreatine and mucosal O2 consumption were studied using in vitro bullfrog gastric mucosa. Electrophysiological parameters (potential difference and resistance) changed rapidly after exposure to 10 or 20 mM acetylsalicylic acid at pH 3.0, 4.0, or 6.0 in the mucosal solution. ATP and phosphocreatine content decreased rapidly after exposure. Control mucosae contained 11.8±0.98 nmoles of ATP and 11.5±0.68 nmoles of phosphocreatine per mg of protein. ATP content decreased 21% and phosphocreatine decreased 45% after 15 to 30 min of exposure. Longer durations of exposure reduced both ATP and phosphocreatine to 10 to 15% of control levels. O2 consumption was increased during the first 15 to 30 min after exposure to acetylsalicylate. Longer durations of exposure reduced O2 consumption to near control levels. Study of isolated mitochondria confirmed that acetylsalicylate itself did not significantly affect mitochondrial respiration, respiratory control ratio, or ADP:O ratio. Salicylate had a biphasic effect which was concentration dependent. Salicylate initially stimulated and later inhibited mitochondrial respiration. Using various substrates the site of inhibition appeared to be located in the segment of the respiratory chain between coenzyme Q and cytochrome c1.