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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-inactivating phenotype. These results strongly suggest that Ca2+ binding to the alpha 1C subunit initiates Ca2+ inactivation.