During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by CaV1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of β-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the α1 subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to β-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective β1- and β2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N′,N′-tetraacetic acid (BAPTA)–acetoxymethyl ester to buffer intracellular calcium results in only β1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to β1-adrenergic receptors. Thus, β-adrenergic stimulation results in phosphorylation of S1928 on the CaV1.2 α1 subunit in intact ventricular myocytes via both β1- and β2-adrenergic receptor pathways, but the β2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with β1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.