1. The properties of the delayed rectifier K+ current (IK) of rabbit isolated sino-atrial node cells were investigated in high (140 mM) [K+]o using the whole-cell-clamp technique. 2. Hyperpolarizing clamp pulses from 0 mV induced an instantaneous current jump (I-V relation linear) followed by a time-dependent increase in inward current to a peak, whereas depolarizing clamp pulses induced little outward current. The peak I-V relation showed a strong inward rectification. The inwardly rectifying current was blocked by E-4031. 3. The inward K+ current induced by hyperpolarizing clamp pulses from 0 mV relaxed after reaching its peak. The rate of the relaxation increased as the membrane potential became more negative and concentrations of external Ca2+ or Mg2+ were increased. The steady-state current was smaller as the relaxation of the current accelerated on increasing [Ca2+]o or [Mg2+]o. 4. Depolarizing clamp pulses from -80 mV induced an increase in inward current, reaching a steady state. The amplitude of the steady-state current became smaller and the rate of current increase became slower as [Ca2+]o or [Mg2+]o was increased. 5. The effects of Ca2+ and Mg2+ are well explained by a time- and voltage-dependent blockade of the K+ channel by these ions. The fractional electrical distance of the binding site calculated from the voltage dependence of the blocking rate constant is 0.69 for Ca2+ and 0.88 for Mg2+. The blocking rate constant at 0 mV for Ca2+ is about 15 times faster than that for Mg2+, indicating stronger effects of Ca2+. 6. A re-interpretation of IK in sino-atrial node cells is proposed: there are two independent gates (an activation gate which opens on hyperpolarization and an inactivation gate which closes on hyperpolarization) and a binding site for Ca2+ and Mg2+ inside the channel. Binding of these ions, which is facilitated by hyperpolarization, causes channel blockade, resulting in the observed voltage dependence of IK in physiological concentrations of Ca2+ and Mg2+.