Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3,5-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase C␣ independently and to the same extent as serine 67, the previous protein kinase C␣-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.A fine-tuned regulation of protein kinase and protein phosphatase activities is essential in the control of the phosphorylation state of various key phosphoprotein substrates, which modulate glycogen metabolism, protein synthesis, cell division, neuronal signaling, and muscle contraction (1-3). A cross-talk between the second messenger cAMP-dependent protein kinase (PKA) 4 and the type-1 protein phosphatase (PP1) occurs at the level of an endogenous phosphoprotein, inhibitor-1 (I-1), allowing amplification of the cAMP signaling cascade. Inhibitor-1 was first identified in rabbit skeletal muscle (4), but it is widely expressed in mammalian tissues and highly conserved across species (5). This thermostable protein, upon phosphorylation by PKA at Thr-35, potently inhibits PP1, enhancing cAMP-mediated protein phosphorylation (6 -9). In cardiac muscle, the regulation of PP1 by I-1 has been shown to play a major role in both basal contractility (2, 10), as well as in the heart response to -adrenergic stimulation (11, 12). The positive inotropic effect of the -agonist, isoproterenol, is accompanied by I-1 phosphorylation, resulting in inhibition of PP1 activity (13). This decrease in PP1 augments the cAMP-stimulating effects in enhancing cardiac contractility by preventing the dephosphorylation of important proteins involved in regulating the contractile state of the heart. Intriguingly, a constitutively activated form of I-1 (T35D; AA1-65) enhanced cardiac function and the heart response to -adrenergic agonists, and protected the heart from developing hypertrophy induced by pressure overload (10). Furthermore, acute adenovira...