Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the β₂ subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat β_2a subunit was phosphorylated at two sites in vitro by purified PKA. This β_2a subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr₁₆₄ and Ser₅₉₁. However, upon mutation of both of these residues to alanines, the β_2a subunit remained a good substrate for PKA. The actual sites of phosphorylation on the β_2a subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed β_2a subunit demonstrated that this subunit was phosphorylated similarly to the β₂ subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the β_2a subunit. The results suggest that phosphorylation of the β_2a subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most β₂ subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated β₂ subunits lacking PKA sites remained good substrates for PKC.