A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.