Recent studies have demonstrated that phosphoinositide 3-kinases (PI3Ks) play a fundamental role in regulating myocardial contractility. However, even though alpha1-adrenergic receptor stimulation is known to activate PI3Ks, the impact of this pathway on the inotropic effects of alpha1-stimulation is unclear. Isolated rabbit ventricular myocytes were preincubated with the PI3K inhibitor wortmannin (WM, 0.1 micromol/L). The alpha1 agonist phenylephrine (PE, 10 micromol/L) induced a significantly stronger increase in contractility in WM-treated versus control myocytes (Fractional shortening in percent of resting cell length: 6.14+/-0.33 percent; n=26 versus 4.85+/-0.33 percent; n=26, P less than 0.05). Furthermore, pretreatment with WM significantly increased the positive inotropic effect of PE in intact muscle strips from rabbit hearts. Mechanistically, we demonstrate that in WM-treated myocytes PE increased phospholamban (PLN) phosphorylation and intracellular Ca2+ transients to a significantly greater extent than in control myocytes. In summary, this is the first study to demonstrate that inhibition of PI3K by increasing PLN phosphorylation and Ca2+ transients significantly improves contractility in alpha1-adrenergically stimulated myocardium. This may have clinical implications for the treatment of decreased cardiac function in acute heart failure.