Calcium influx through the voltage-dependent L-type calcium channel (Ca_V1.2) rapidly increases in the heart during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca_V1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α_1C and C-terminal proteolytic cleavage of the α_1C subunit. We generated transgenic mice expressing an α_1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca_V1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α_1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α_1C. Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca_V1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α_1C in β-adrenergic stimulation of Ca_V1.2, and show that phosphoregulatory sites on α_1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α_1C is not required for β-adrenergic stimulation of Ca_V1.2.