We previously showed that palmitoyl-L-carnitine (L-PC) inhibits the Na/K pump current (INa/K). In the present report, we test the hypothesis that L-PC, like ouabain, should increase myocyte shortening. Membrane potentials or ionic currents were recorded simultaneously with cell shortening in single guinea pig ventricular myocytes at room temperature (22 degrees C). Like ouabain, L-PC (1 microM) reversibly depolarized the resting membrane, decreased action potential duration, and increased the amplitude of myocyte contractions. Neither L-PC nor ouabain had a significant effect on Ca current (ICa). When L-PC increased cell shortening during ramp voltage clamp, membrane current shifted inward at voltages negative to -20 mV and shifted outward at more positive voltages. Similar to toxic concentrations of ouabain, L-PC induced transient inward currents and aftercontractions. At concentrations that inhibit INa/K, L-PC acted like ouabain to produce characteristic effects on membrane potentials, currents, and cell contractions that were unrelated to significant changes in ICa. L-PC reduces surface negative charge of erythrocytes and myocytes (C. Gruver and A. J. Pappano, J. Mol. Cell. Cardiol. 25: 1275-1284, 1993), and we speculate that L-PC inhibits INa/K by this mechanism.