The loci for inactivation in calcium channel proteins are unknown. Mechanisms for inactivation may be distributed across Ca2+ channel subunits and appear to be complex, multiple and interacting. We took advantage of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel alpha 1 subunits to study the molecular mechanism of inactivation in L-type calcium channels. Sk1H3, a chimeric construct of these two L-type calcium channels, was expressed in Xenopus oocytes in the absence of auxiliary subunits. Sk1H3 incorporated repeat I from skeletal muscle alpha 1 and repeats II, III, IV from heart alpha 1 subunit. Sk1H3 inactivated faster (tau = 300 ms) and more fully than the wild-type H4 with Ba2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete after a 5-s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative potentials with E0.5 = -15 mV as compared to E0.5 = -5 mV for H4. In the presence of external calcium ions, the extent of inactivation significantly increased from 37 to 83% for H4 while inactivation of Sk1H3 was only slightly increased. Inactivation with Ba2+ as the charge carrier was confirmed at the single- channel level where averaged single-channel ensembles showed a similar rate of inactivation. Collectively, these observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage-dependent component. Whether Sk1H3 inactivation involves interactions within repeat I alone or interactions between repeat I and site(s) located in the three other repeats of the alpha 1 subunit has yet to be determined.