During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca²⁺) currents conducted by Ca_V1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′-monophosphate–dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca_V1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca_V1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α₂δ₁ and β_2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca_V1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca_V1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca_V1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.