Stimulation of cardiac -adrenergic receptors (AR) activates both the G_s- and G_i-coupled signaling cascades, including the PI3K pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Co-treatment with ICI 118,551, but not CGP 20712, abolished the increase in PI3K activity, suggesting a 2AR-mediated event. PI3K activation was also abrogated by co-treatment with pertussis toxin, PP2 (selective Src-family tyrosine kinases inhibitor), or AG1296 (selective PDGFR inhibitor) but not with an inhibitor for PKA, PKC, Ras, adenylyl cyclase, EGFR or IGF-1R. AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or siRNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a G sequestrant peptide derived from the C-terminus of AR kinase-1 failed to activate PI3K after AR stimulation, suggesting G is required for the transactivation. Furthermore, acute AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3K isoform activities but not the activities of other isoforms (PI3K, -, -) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of AR-mediated transactivation of cardiac PI3K via sequential involvement of G_i/G, Src and PDGFR.