Phospholamban is a critical regulator of sarcoplasmic reticulum Ca²⁺-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca²⁺ and cAMP pathways, we have investigated the β-adrenergic-induced phosphorylation of Ser¹⁶ and Thr¹⁷ of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser¹⁶ and Thr¹⁷ with strikingly different half-maximal values (EC₅₀ = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser¹⁶ induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr¹⁷ did not. Elevation of extracellular Ca²⁺did not induce phosphorylation at Thr¹⁷; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr¹⁷ increased eightfold without additional effects on relaxation rate. Thr¹⁷ phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 μmol/l verapamil or nifedipine. The data indicate that1) phosphorylation of phospholamban at Ser¹⁶ by cAMP-dependent protein kinase is the main regulator of β-adrenergic-induced cardiac relaxation definitely preceding Thr¹⁷ phosphorylation and2) the β-adrenergic-mediated phosphorylation of Thr¹⁷ by Ca²⁺-calmodulin-dependent protein kinase required influx of Ca²⁺through the L-type Ca²⁺ channel.