Treatment of fibroblasts with wortmannin or demethoxyviridin, two potent inhibitors of phosphatidylinositol 3-kinase, prevents the activation of ribosomal protein S6 kinase, which is induced by a variety of external stimuli. Concentrations giving 50% inhibition of 45 nM (wortmannin) and 400 nM (demethoxyviridin) were obtained when epidermal growth factor was used as an S6 kinase activator; with platelet-derived growth factor, the concentrations giving 50% inhibition were about three-times higher. Western-blot analysis and immunocomplex kinase assays showed that wortmannin and demethoxyviridin specifically block the phosphorylation and activation of p70 S6 kinase without affecting the M(r) 90,000 ribosomal S6 kinase (p90rsk) or mitogen-activated protein kinases. Consistent with the irreversible nature of the inhibition of phosphatidylinositol 3-kinase by these compounds, treatment of cells with wortmannin, followed by washing out of the inhibitor, still led to inhibition of p70 S6 kinase activation. Several S6 kinase agonists not previously known to activate phosphatidylinositol 3-kinase (A23187, bombesin and phorbol 12-myristate 13-acetate) were found to increase the production of phosphatidylinositol 3,4,5-trisphosphate in a wortmannin-sensitive manner. These results support a model in which phosphatidylinositol 3-kinase acts upstream of p70 S6 kinase in a mitogenic signalling pathway; the existence of a phosphatidylinositol 3-kinase-independent pathway is also evident.