The benzothiazepine diltiazem blocks ionic current through L-type Ca(2+) channels, as do the dihydropyridines (DHPs) and phenylalkylamines (PAs), but it has unique properties that distinguish it from these other drug classes. Wild-type L-type channels containing alpha(1CII) subunits, wild-type P/Q-type channels containing alpha(1A) subunits, and mutants of both channel types were transiently expressed in tsA-201 cells with beta(1B) and alpha(2)delta subunits. Whole-cell, voltage-clamp recordings showed that diltiazem blocks L-type Ca(2+) channels approximately 5-fold more potently than it does P/Q-type channels. Diltiazem blocked a mutant P/Q-type channel containing nine amino acid changes that made it highly sensitive to DHPs, with the same potency as L-type channels. Thus, amino acids specific to the L-type channel that confer DHP sensitivity in an alpha(1A) background also increase sensitivity to diltiazem. Analysis of single amino acid mutations in domains IIIS6 and IVS6 of alpha(1CII) subunits confirmed the role of these L-type-specific amino acid residues in diltiazem block, and also indicated that Y1152 of alpha(1CII), an amino acid critical to both DHP and PA block, does not play a role in diltiazem block. Furthermore, T1039 and Y1043 in domain IIIS5, which are both critical for DHP block, are not involved in block by diltiazem. Conversely, three amino acid residues (I1150, M1160, and I1460) contribute to diltiazem block but have not been shown to affect DHP or PA block. Thus, binding of diltiazem to L-type Ca(2+) channels requires residues that overlap those that are critical for DHP and PA block as well as residues unique to diltiazem.