The goal of this study was to determine if the protein kinase A (PKA)-responsiveness of the cardiac L-type Ca²⁺ current (I_Ca) is affected during transient increases in the intracellular Ca²⁺ concentration. Ventricular myocytes were isolated from 3-4 day old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca²⁺ external solution, the aligned myocytes displayed a large I_Ca that was weakly regulated (20 % increase) during stimulation of PKA by 2 µM forskolin. In contrast, application of forskolin caused a 100% increase in I_Ca when the external Ca²⁺ concentration was reduced to 0.5 mM or replaced with Ba²⁺. This Ca²⁺-dependent inhibition was also observed when the cells were treated with isoproterenol (1 µM), 3-isobutyl-1-methylxanthine (100 µM) and 8-bromo cAMP (500 µM). The responsiveness of I_Ca to PKA was restored during intracellular dialysis with a calmodulin inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca²⁺/calmodulin-dependent protein kinase or calcineurin. Adenoviral-mediated expression of a calmodulin molecule with mutations in all four Ca²⁺ binding sites, also increased the PKA-sensitivity of I_Ca. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba²⁺. Thus, Ca²⁺ entering into the myocyte through the voltage-gated Ca²⁺ channel regulates the PKA-responsiveness of I_Ca.