The effects of aspirin on epithelial cell membrane potentials of Necturus fundic mucosa were examined by in vitro experiments according to intracellular microelectrode techniques. Stable intracellular impalements were obtained with 15 to 50 M-ohm microelectrodes filled with 3M KCl. In neutral mucosal solutions (pH 7.0) aspirin (5.0 mM) resulted in a significant increase in apical cell membrane potential (Vmc) from -36.7 +/- 1.5 mV to -43.3 +/- 2.3 mV (p less than 0.001) and basolateral cell membrane potential (Vcs) from -42.7 +/- 1.8 mV to -50.6 +/- 2.4 (p less than 0.001). This hyperpolarization of the cell was associated with an increase in transmucosal potential from -5.8 +/- 0.7 to -7.4 +/- 0.9 (p less than 0.05) and an increase in the ratio of apical to basolateral membrane resistances from 5.1 +/- 1.2 to 8.8 +/- 1.9 (p less than 0.05). These changes were consistent with an increase in potassium conductance induced by the salicylate anion. In acidic mucosal solutions (pH 4.5) aspirin caused a reduction in Vmc and Vcs. This hypopolarization of the cell membrane is consistent with acidification of the epithelial cells. These observations support the proposed mechanisms of aspirin injury: (1) back diffusion of H+ into the cells and (2) influx of the salicylate anions into the cells, which may interfere with intracellular metabolism.