1. The block of the inwardly rectifying K+ channel by intracellular Ca2+ was studied in guinea-pig ventricular cells. 2. Single-channel currents through the inwardly rectifying K+ channel were recorded in the inside-out configuration at 150 mM external and internal K+. Internal Ca2+, at a concentration of 0.4-10 microM, induced subconductance levels with one-third and two-thirds of the unitary amplitude in the outward currents without affecting the inward currents. 3. Occupancy at each sublevel was estimated from the amplitude histogram which showed four equally spaced peaks in the presence of internal Ca2+. At different degrees of blockade, the distribution of the current levels showed a reasonable agreement with the binomial theorem. 4. The outward mean open-channel currents were measured at different Ca2+ concentrations and voltages. The current-voltage relation rectified inwardly in the presence of internal Ca2+ in a concentration-dependent manner. 5. The outward mean open-channel currents were normalized to unitary amplitudes in the absence of Ca2+. The normalized current-Ca2+ concentration curve was fitted by saturation kinetics with a Hill coefficient of 1 at each voltage. The voltage dependence of the dissociation constants gives the value for the fractional electrical distance of the Ca2+ binding site of 0.7. 6. The dwell times in each substrate were distributed exponentially. On the assumption that the inwardly rectifying K+ channel of cardiac cells is composed of three identical conducting subunits and each subunit is blocked by Ca2+ independently, the blocking (mu) and unblocking (lambda) rates were calculated. The value of mu increased with higher Ca2+ concentrations or larger depolarizations, while lambda was independent of Ca2+ and decreased with larger depolarization. 7. It is thus concluded that internal Ca2+ produces a voltage-dependent block of the channel to cause inward rectification although the blocking effect is less potent than that of Mg2+. The substate behaviour seen with internal Ca2+ supports the triple-barrelled structure of the cardiac inwardly rectifying K+ channel.