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Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3Kalpha, -beta, and -delta isoforms coupled to receptor tyrosine kinases and a PI3Kgamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3Kgamma catalytic subunit (p110gamma) and Gbetagamma complexes. When phosphatidylinositol was used as a substrate, Gbetagamma stimulated p110gamma lipid kinase activity more than 60-fold (EC50, approximately 20 nM). Stimulation was inhibited by Galphao-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110gamma did not affect Gbetagamma-mediated enzymatic stimulation, whereas incubation of Gbetagamma with a synthetic peptide resembling a predicted Gbetagamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110gamma. Gbetagamma complexes bound to N- as well as C-terminal deletion mutants of p110gamma. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited Gbetagamma-mediated activation of wild type p110gamma. Our data suggest that (i) p110gamma directly interacts with Gbetagamma, (ii) the pleckstrin homology domain is not the only region important for Gbetagamma-mediated activation of the lipid kinase, and (iii) Gbetagamma binds to at least two contact sites of p110gamma, one of which is close to or within the catalytic core of the enzyme.