The epidermal growth factor receptor (EGFR) is regulated by at least two mechanisms involving protein kinase C (PKC), inhibition of EGF binding and inhibition of EGF-stimulated tyrosine kinase activity. In this study we investigated whether mitogen-activated protein kinase (MAPK) mediates the inhibitory effects of PKC on EGFR binding or kinase activity by pretreating NIH3T3 and Chinese hamster ovary cells expressing the EGFR with PD98059, an inhibitor of MAPK/extracellular signal-regulated kinase kinase (MEK). We also determined whether substitution of cysteine for threonine at residue 669, the site of MAPK phosphorylation of the EGFR, alters the inhibition of kinase activity by PKC. The results indicate that 1) PKC down-regulates EGFR tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; 2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; 3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and 4) PKC-mediated down-regulation of EGF binding and EGFR kinase activity occur by different mechanisms. These data are consistent with a model for regulation of the EGFR by other receptors whereby their activation of PKC, in conjunction with MAPK, results in the phosphorylation of a protein(s) that modulates EGFR kinase activity.