The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.