Treatment of A431 cell membranes with trypsin or Streptomyces griseus proteinase results in degradation of the EGF-R and the concomitant generation of an active kinase with a molecular mass of 42 kDa (42 kDa kinase). To investigate the biochemical properties of the 42 kDa kinase, the EGF-R was immunoaffinity-purified from the A431 cell membranes and the kinase proteolytically generated. The proteolysis of EGF-R changes both the Vmax and the Michaelis constants of substrates. These substrates determine the extent of the changes of the parameters. The 42 kDa kinase is less responsive to polyions as regulators of kinase activity and is less efficiently inhibited by genistein and tyrphostin. The experiments described here point to a role of the extracatalytic domains in determining the substrate specificity and regulation of kinase activity.