The regulation of Ca²⁺ influx through the phosphorylation of the L-type Ca²⁺ channel, Ca_v1.2, is important for the modulation of excitation−contraction (E−C) coupling in the heart. Ca_v1.2 is thought to be the target of multiple kinases that mediate the signals of both the renin-angiotensin and sympathetic nervous systems. Detailed biochemical information regarding the protein phosphorylation reactions involved in the regulation of Ca_v1.2 is limited. The protein kinase C (PKC) family of kinases can modulate cardiac contractility in a complex manner, such that contractility is either enhanced or depressed and relaxation is either accelerated or slowed. We have previously reported that Ser¹⁹²⁸ in the C-terminus of α_1c was a target for PKCα, -ζ, and -ε phosphorylation. Here, we report the identification of seven PKC phosphorylation sites within the α_1c subunit. Using phospho-epitope specific antibodies to Ser¹⁶⁷⁴ and Ser¹⁹²⁸, we demonstrate that both sites within the C-terminus are phosphorylated in HEK cells in response to PMA. Phosphorylation was inhibited with a PKC inhibitor, bisindolylmaleimide. In Langendorff-perfused rat hearts, both Ser¹⁶⁷⁴ and Ser¹⁹²⁸ were phosphorylated in response to PMA. Phosphorylation of Ser¹⁶⁷⁴, but not Ser¹⁹²⁸, is PKC isoform specific, as only PKCα, -βI, -βII, -γ, -δ, and -θ, but not PKCε, -ζ, and -η, were able to phosphorylate this site. Our results identify a molecular mechanism by which PKC isoforms can have different effects on channel activity by phosphorylating different residues.