The time course of the inward-rectifying K current during hyperpolarizing clamp steps was investigated in single myocytes isolated from guinea-pig ventricles. The experiments were done using a two-electrode voltage-clamp technique with two patch pipettes in the whole-cell configuration. Hyperpolarizations to potentials negative to -100 mV, induced large inward-rectifying K currents (iK1), which showed a marked decay. The current-voltage relation of the peak inward current was almost linear, but the steady-state current-voltage relation had a region of negative slope at potentials negative to -140 mV. These findings indicate that the channel inactivates during hyperpolarizing steps. When Na ions in the extracellular solution were replaced by choline, Tris, TMA or sucrose, the decay of the inward currents was largely reduced, and the negative slope in the steady-state current-voltage relation was absent. When divalent ions were removed from the Na-free bathing solution, a marked increase in iK1 was found, and the currents became time-independent. These experiments demonstrate that the inactivation during hyperpolarization is largely due to a block of the channel by external Na ions. The block by Na is most pronounced at very negative potentials, and is strongly voltage-dependent. External Ca and Mg ions also cause a marked block of the channel. The block by these divalent ions is however much less voltage-dependent than the one by Na, but is already present at the cell's resting potential.