The maximal rates of the protein kinase (ATP: protein phosphotransferase, EC 18.104.22.168) reaction studied with chicken egg yolk phosvitin as substrate are dependent on the level of dephosphorylation of phosvitin. 30% dephospho-phosvitin gives the optimal initial rates. With varying levels of dephosphorylation, the apparent Km for the substrate also changes in a biphasic manner. If this factor is taken into account, and a suitable adjustment is made for the concentration of dephospho-phosvitin in the reaction, it is possible to achieve maximal rates for the kinase reaction with phosvitin preparations of varying levels of dephosphorylation. Such a consideration is important for comparing the results of protein kinase studies using phosvitin as the substrate.