Recent findings indicate that platelet-derived growth factor (PDGF) plays a role in the generation of reactive oxygen species (ROS) as second messengers in smooth muscle cells (SMC). To identify the source and signal transduction pathway of ROS formation in SMC, we investigated PDGF-induced ROS formation. Stimulation of SMC with PDGF resulted in a rapid increase of ROS production. Using an inactivating antibody, we identified the increase to be dependent on p22phox, a NAD(P)H-oxidase subunit. ROS release was completely inhibited by the Gi protein inhibitor PTX as well as an antibody against Galphai1,2, however, not by antibodies against Galphai3/0, Gas, and Gbeta1beta2. The effect of PDGF on ROS production in SMC membranes could likewise be mimicked by the use of a recombinant Galphai2 subunit but not by Galphai3, Galphai0, Gas, and Gbetagamma subunits. Immunoaffinity chromatography demonstrated coupling of Galphai1,2 to the PDGF a-receptor, which, after preincubation of the SMC membranes with PDGF, was increased in the absence of GTPgammaS but decreased in the presence of GTPgammaS and prevented by PTX treatment. These data define a novel G protein-dependent mechanism by which PDGF signaling is transduced through direct coupling of the Gai1,2 subunit of the trimeric G proteins to the PDGF tyrosine kinase receptor.