VOLTAGE-DEPENDENT Ca2+ channels respond to membrane depolarization by conformational changes that control channel opening and eventual closing by inactivation(1-3). The kinetics of inactivation differ considerably between types of Ca2+ channels(1-8) and are important in determining the amount of Ca2+ entry during electrical activity and its resulting impact on diverse cellular events(3). The most intensively characterized forms of inactivation in potassium(9,10) and sodium channels(11-13) involve pore block by a tethered plug(14). In contrast, little is known about the molecular basis of Ca2+-channel inactivation. We studied the molecular mechanism of inactivation of voltage-gated calcium channels by making chimaeras from channels with different inactivation rates. We report here that the amino acids responsible for the kinetic differences are localized to membrane-spanning segment S6 of the first repeat of the alpha(1) subunit (IS6), and to putative extracellular and cytoplasmic domains flanking IS6. Involvement of this region in Ca2+-channel inactivation was unexpected and raises interesting comparisons with Na+ channels, where the III-IV loop is a critical structural determinant. Ca2+-channel inactivation has some features that resemble C-type inactivation of potassium channels.