Tyrosine phosphorylation of a 42-kD, cytosolic protein is a rapid consequence when quiescent cells are stimulated with any one of a diverse group of mitogenic agents. Among the inducers of this tyrosine phosphorylation are activators of protein kinase C, raising the possibility that this serine/threonine-specific protein kinase plays a role in mitogen-induced tyrosine phosphorylation. Using fibroblastic cells depleted of protein kinase C by chronic treatment with the tumor promoter tetradecanoyl phorbol acetate (TPA), we now show that protein kinase C is required for the tyrosine phosphorylation of the 42-kD protein, even when epidermal growth factor (EGF), whose receptor is a tyrosine-specific protein kinase, provides the initial stimulus. EGF is able to induce other cellular phosphorylations independent of protein kinase C, whereas thrombin appears to require the protein kinase C-dependent pathway. These findings suggest that phosphorylation of the 42-kD protein is part of a protein kinase C-dependent kinase cascade involved in intracellular signalling.