Neuropeptide-stimulated tyrosine phosphorylation of specific components in Swiss 3T3 cells was investigated using monoclonal antibodies directed against the src transformation-associated substrates p125 focal adhesion kinase (FAK), a novel type of cytosolic tyrosine kinase, and p130. Treatment of Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, and endothelin caused a striking increase in the tyrosine phosphorylation of p125FAK, as judged either by anti-phosphotyrosine (anti-Tyr(P)) Western blots of anti-p125FAK immunoprecipitates, or by anti-p125FAK immunoblots of anti-Tyr(P) immunoprecipitates. Bombesin-stimulated tyrosine phosphorylation of p125FAK was detectable within seconds and concentration-dependent (half-maximum effect of 0.3 nM). Neuropeptides also stimulated the tyrosine phosphorylation of a second component of M(r) 130,000, previously identified as the major p130 phosphotyrosyl protein in src-transformed cells. Bombesin stimulated p130 tyrosine phosphorylation with kinetics and concentration dependence similar to those observed for p125FAK. This is the first report to identify substrates for neuropeptide-stimulated tyrosine phosphorylation; the finding that one of these substrates is a tyrosine kinase suggests the existence of a novel signal transduction pathway in the action of mitogenic neuropeptides.