Integrin adhesion to extracellular matrix proteins protects adhesion-dependent cells from suspension-induced apoptosis. Previous studies indicate that activation of the transcription factor nuclear factor-kappaB was necessary for the integrin alpha(v)beta3 ligand osteopontin to protect endothelial cells from apoptosis caused by serum withdrawal. In this study, beta3 integrins were overexpressed in smooth muscle cells. When plated on osteopontin, cells overexpressing wild-type beta3 had enhanced cell adhesion, cell spreading, and nuclear factor-kappaB activation compared with vector control. Removal of four amino acids (759X) from the C terminus of beta3 eliminated the ability of the integrin to promote these processes. Single amino acid substitutions indicated that phosphorylation at tyrosine 759 was not required for activation of the transcription factor, however this residue appeared to play a structural role, because mutation to alanine significantly inhibited nuclear factor-kappaB activation. The Src family of tyrosine kinases represents important transducers during integrin signaling, and the C terminus of beta3 has been implicated as the binding site for Src. Immunoprecipitations demonstrated that Src associated with wild-type beta3 integrins, but Src and integrins lacking the C terminus (759X) did not form a complex. Pharmacological inhibition with the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) or overexpression of kinase-dead c-Src blocked nuclear factor-kappaB activation. Mouse embryonic fibroblasts deficient for Src failed to activate nuclear factor-kappaB when plated on osteopontin, in contrast to control fibroblasts. Together, these experiments indicate that the C terminus of beta3 and Src activity are required for integrin alpha(v)beta3-mediated nuclear factor-kappaB activation.