Dephosphorylated phospholamban (PLB) is an inhibitor of the affinity of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2) for Ca2+. Phosphorylation of PLB relieves its inhibitory effects on SERCA2, with subsequent acceleration of Ca2+ transport into the SR lumen, which has been suggested to underlie the positive inotropic and lusitropic actions of beta-adrenergic agonists in the mammalian heart. PLB can be phosphorylated at Ser16 by cAMP-dependent protein kinase (PKA) and Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII) during beta-agonist stimulation. However, the interrelationship and relative contribution of dual site phosphorylation to the cardiac stimulatory effects are not clear. The recent availability of the PLB knockout mouse, in combination with mutagenesis and transgenic technologies, have provided excellent model systems for expression of each of the phosphorylation site-specific PLB mutants in the heart and elucidation of the functional interplay between PKA- and CaMKII-dependent pathways of PLB phosphorylation. Transgenic mice expressing similar levels of the wild-type, S16A, or T17A mutant PLB in the null background were generated and they were characterized in parallel. Our results indicate that 1) reinsertion of PLB into the knockout mouse heart reverses the hyperdynamic cardiac function associated with PLB deficiency, 2) phosphorylation of Ser16 in PLB is sufficient to mediate its maximal cardiac contractile responses to beta-adrenergic stimulation, and 3) Ser16 phosphorylation is a prerequisite for Thr17 phosphorylation in vivo during beta-agonist stimulation, but Thr17 can be phosphorylated independently of Ser16 in vitro. Thus, these studies revealed novel insights into the interdependence and physiological significance of PKA (Ser16) and CaMKII (Thr17) pathways of PLB phosphorylation during beta-adrenergic stimulation in the mammalian heart.