Voltage-gated L-type Ca²⁺ channels mediate Ca²⁺ entry into cells in response to membrane depolarization. Ca²⁺ entry through the cardiac Ca²⁺ channel determines the rate and force of contraction, and modulation of Ca²⁺ channel activity by β-adrenergic agents acting through adenosine 3‘,5‘-cyclic monophosphate- (cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the α1 subunit of the cardiac L-type Ca²⁺ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call α1₂₄₂ and α1₂₁₀. α1₂₄₂ corresponds to the full-length cardiac α1 subunit predicted from its cDNA sequence, while α1₂₁₀ is truncated at its COOH terminus. Only α1₂₄₂ is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that α1₂₄₂ is phosphorylated in intact cells expressing the cardiac α1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the α1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.