Stimulation of fibroblasts with basic fibroblast growth factor (bFGF) led to the rapid tyrosine phosphorylation of a number of cellular proteins, including a major substrate of 90 kDa. The methyltransferase inhibitor 5'-methylthioadenosine (MTA) was found to be a specific inhibitor of bFGF-stimulated protein tyrosine phosphorylation in fibroblasts, blocking both receptor autophosphorylation and substrate phosphorylation. MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in fibroblasts. MTA also inhibited both bFGF-stimulated protein tyrosine phosphorylation and neurite outgrowth in PC12 cells. MTA was a specific inhibitor of bFGF-stimulated protein tyrosine phosphorylation only in intact cells. MTA delayed and reduced, but did not inhibit, bFGF internalization and processing. The effects of MTA on bFGF-stimulated tyrosine phosphorylation required only a brief pretreatment with the agent and were readily reversible.