Compartmentation of cAMP is thought to generate the specificity of G_s-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca²⁺ current (I_Ca,L) on stimulation with β₁-adrenergic receptors (β₁-ARs), β₂-ARs, glucagon receptors (Glu-Rs) and prostaglandin E₁ receptors (PGE₁-Rs). All receptors but PGE₁-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide–gated (CNG) channels, stimulation of β₁-AR and Glu-R increased cAMP, whereas β₂-AR and PGE₁-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated β₁-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE₁-R and β₂-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE₁-R, the measurements of I_Ca,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled β₁-AR and β₂-AR regulation of I_Ca,L, whereas only PDE4 controlled Glu-R regulation of I_Ca,L thus demonstrating that receptor–PDE coupling has functional implications downstream of cAMP. PGE₁ had no effect on I_Ca,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE₁ to diffuse to L-type Ca²⁺ channels. These results identify specific functional coupling of individual PDE families to G_s-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.