The tyrosine kinase class of receptors induces mitogen-activated protein kinase (MAPK) activation through the sequential interaction of the signaling proteins Grb2, Sos, Ras, Raf, and MEK. Receptors coupled to heterotrimeric guanine triphosphate-binding protein (G protein) stimulate MAPK through G(beta gamma) subunits, but the subsequent intervening molecules are still poorly defined. Overexpression of phosphoinositide 3-kinase gamma (PI3K gamma) in COS-7 cells activated MAPK in a G(beta gamma)-dependent fashion, and expression of a catalytically inactive mutant of PI3K gamma abolished the stimulation of MAPK by G(beta gamma) or in response to stimulation of muscarinic (m2) G protein-coupled receptors. Signaling from PI3K gamma to MAPK appears to require a tyrosine kinase, Shc, Grb2, Sos, Ras, and Raf. These findings indicate that PI3K gamma mediates G(beta gamma)-dependent regulation of the MAPK signaling pathway.