Intracellular calcium (Ca2+) inhibits the opening of L-type (alpha(1C)) Ca2+ channels, providing physiological control of Ca2+ entry into a wide variety of cells. A structural determinant of this Ca2+-sensitive inactivation was revealed by chimeric Ca2+ channels derived from parental alpha(1C) and alpha(1E) channels, the latter of which is a neuronal channel lacking Ca2+ inactivation. A consensus Ca2+-binding motif (an EF hand), located on the alpha(1C) subunit, was required for Ca2+ inactivation. Donation of the alpha(1C) EF-hand region to the alpha(1E) channel conferred the Ca2+-inactivating phenotype. These results strongly suggest that Ca2+ binding to the alpha(1C) subunit initiates Ca2+ inactivation.