It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage-dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (tau approximately 6 s, 21 degrees C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (tau approximately 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca(2+) -dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and F., Ríos. 1993. J. Gen. Physiol. 102:1005-1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the alpha 1 subunit to be 100 times lower for Ba2+ than Ca2+.