The PI3K signaling pathway regulates multiple cellular processes including cell survival/apoptosis and growth. In the cardiac context, PI3K plays important roles in cardiac growth. We have shown that cardiac PI3K activity is highly regulated during development, with the highest levels found during the fetal-neonatal transition period and the lowest levels in the adult. There is a close relationship between cardiomyocyte proliferation and cardiac PI3K activity. In adult transgenic mice, however, prolonged constitutively activation of PI3K in the heart results in hypertrophy. To develop a strategy to allow temporally controlled overexpression of cardiac PI3K, we engineered a tetracycline (tet) transactivator (tTA) tet-off controlled transgenic mouse line with conditional overexpression of a cardiac-specific fusion protein of the SH2 domain of p85 and p110. Cardiac PI3K activity and Akt phosphorylation were significantly increased in adult mice after transgene induction following removal of doxycycline for 2 weeks. Heart weight to body weight ratio was not changed and there were no signs of cardiomyopathy. Overexpression of PI3K resulted in increased in left ventricular (LV) developed pressure, LV dP/dt max and LV dP/dt min, but not heart rate, as assessed in Langendorff hearts. Mice overexpressing PI3K also had increases in the levels of Ca²⁺-regulating proteins, including the L-type Ca²⁺ channels, ryanodine receptors, and SERCA2a. Thus, temporally controlled overexpression of cardiac PI3K does not induce hypertrophy or cardiomyopathy but results in increased contractility, probably via increased expression of multiple Ca²⁺-regulating proteins. These distinct phenotypes suggest a fundamental difference between transgenic mice with temporal or prolonged activation of cardiac PI3K.