We investigated the nature and structural requirements for Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channel. Investigation of subunit requirements indicates that the interaction of alpha 1 subunit with ancillary subunits, especially beta subunit, is important for this property. Replacement of the putative cytoplasmic regions of the cardiac alpha 1 subunit with skeletal muscle counterparts eliminates Ca(2+)-dependent inactivation, indicating that the site regulated by Ca2+ resides in the cytoplasmic region of the alpha 1 subunit. Deletion of the carboxy-terminal region of the cardiac alpha 1 subunit does not eliminate this property, suggesting that the modulation by protein kinase A may not be involved in this mechanism. Single amino acid substitution that strongly reduces Ca2+ selectivity of Ca2+ channels also eliminates Ca(2+)-dependent inactivation, suggesting the close link between the ion selectivity and Ca(2+)-dependent inactivation.