T heophylline enhances the force of diaphragmatic contraction and delays fatigue. The mechanism is not known, but recent evidence suggests it may act at the cell membrane. To test this hypothesis, we studied the effect of theophylline on resting membrane potential and tension in hamster diaphragm cells. Muscle strips were obtained from five adult hamsters and placed in Krebs solution, aerated with 95% O2, 5% CO2. Resting membrane potential was measured using 3-M KCl-filled glass microelectrodes; 15-22 fibers in each strip were sampled. Force frequency curves (twitch to 100 Hz) were obtained. The muscle bath was then changed to one containing 100 mg/liter (0.55) theophylline. Resting membrane potential was -76 +/- 3 mV (mean +/- S.D.) in Krebs solution and increased to -85 +/- 3 mV (P less than 0.01) with added theophylline. Tension increased from 5% (at 100 Hz) to 20% (at 10 Hz) with theophylline. Hyperpolarization indicates an increase in intracellular to extracellular potassium concentration. Net potassium outflow occurs with each contraction, causing the cell membrane to become depolarized with repeated contractions, ultimately leading to fatigue. The hyperpolarization of the skeletal muscle cell membrane observed with theophylline may play an important role in prolonging time to fatigue.