Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microinjected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGF-stimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell viability. Injection of an antibody specific for Src, Fyn, and Yes also reduced signal transduction through the PDGF receptor but only when injected within 8 hr of PDGF stimulation. Together these results indicate that the ubiquitously expressed Src family members are required for PDGF-induced mitogenic signaling.