Contractility and relaxation measurements were combined with the determination of total phospholamban (PLB) phosphorylation and the immunodetection of PLB-phosphorylation sites in the intact, beating rat heart to identify the contributions of PLB phosphorylation at the Thr(17) and Ser(16) residues at different levels of beta-adrenoceptor stimulation. Whereas with 30-300 nM isoproterenol, phosphorylation of Thr(17), the Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) site and Ser(16), the protein kinase A (PKA) site, contributed approximately 50% each to PLB phosphorylation, and both participated in the relaxant action of isoproterenol, at lower a level of beta-adrenoceptor stimulation (isoproterenol 0.3-3 nM), both effects were exclusively due to Ser(16) phosphorylation. Increasing [Ca](o) at 3 nM isoproterenol, to obtain an increase in contractility comparable to that produced by 30 nM isoproterenol, significantly increased Thr(17) phosphorylation and the relaxant effect produced by 3 nM isoproterenol. An increase in Thr(17) phosphorylation and in the relaxant effect of 3 nM isoproterenol was also obtained by phosphatase inhibition (okadaic acid). In this case, Ser(16) phosphorylation was also increased. Moreover, perfusion with 30 nM isoproterenol in the presence of the PKA inhibitor H-89 decreased phosphorylation at both PLB residues and diminished the inotropic and relaxant responses to the beta-agonist. The relative contribution of Thr(17) phosphorylation to the isoproterenol-induced phosphorylation of PLB and relaxation thus increased with the level of beta-adrenoceptor stimulation and the consequent increase in PKA activity. The lack of Thr(17) phosphorylation at low isoproterenol concentrations might therefore be attributed to a level of PKA activity insufficient to increase [Ca](i) to activate the CaMKII system and/or to inhibit the phosphatase that dephosphorylates PLB