Vinexin, a novel protein that plays a key role in cell spreading and cytoskeletal organization, contains three SH3 domains and binds to vinculin through its first and second SH3 domains. We show here that the third SH3 domain binds to Sos, a guanine nucleotide exchange factor for Ras and Rac, both in vitro and in vivo. Point mutations in the third SH3 domain abolished the vinexin-Sos interaction. Stimulation of NIH/3T3 cells with serum, epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) decreased the electrophoretic mobility of Sos and concomitantly inhibited formation of the vinexin-Sos complex. Phosphatase treatment of lysates restored the binding of Sos to vinexin, suggesting that signaling from serum, EGF, or PDGF regulates the vinexin-Sos complex through the Sos phosphorylation. To evaluate the function of vinexin downstream of growth factors, we examined the effects of wild-type and mutant vinexin expression on extracellular signal-regulated kinase (Erk) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation in response to EGF. Exogenous expression of vinexin beta in NIH/3T3 cells enhanced JNK/SAPK activation but did not affect Erk activation. Moreover mutations in the third SH3 domain abolished EGF activation of JNK/SAPK in a dominant-negative fashion, whereas they slightly stimulated Erk. Together these results suggest that vinexin can selectively modulate EGF-induced signal transduction pathways leading to JNK/SAPK kinase activation.