The effects of hypertonic solution on action potential and input resistance were studied using the guinea-pig ventricular muscle. Hypertonic solutions containing excess (150 and 300mOsm/liter) sucrose, glucose, NaCl, Na₂SO₄, or LiCl produced a prolongation of action potential duration (APD) followed by a gradual shortening. These solutions increased the input resistance of the preparation. The prolongation of APD began immediately after the onset of perfusion with hypertonic solution and peaked within about 10min, while the late shortening of APD developed progressively. On introducing the isotonic solution after the osmotic challenge, APD initially shortened and then gradually prolonged toward the control level. Slightly hypertonic solutions containing excess 30 or 75mM sucrose prolonged APD, to some extent. It is concluded that the hypertonic solution exerts early and late effects on APD in guinea-pig ventricular muscle. The early effect, which is responsible for the APD prolongation, seems to be directly related to the development of cell dehydration. The hypertonic solutions produced a slight increase in the resting potential, and sucrose- and glucose-hypertonicity depressed the rising phase of the action potential. These effects can be explained by the cell dehydration and associated changes in the intracellular ion concentrations. The increase in input resistance produced by hypertonic solutions was accompanied by an enhanced spatial decay of electrotonic potentials along the muscle bundle, as determined by the two microelectrode method, suggesting that the electrical resistance of the internal current pathway increases under hypertonic conditions.