The calcium/phospholipid-dependent protein kinase (PKC) and the H4 protease-activated protein kinase (H4PK) from lymphosarcoma cells were separated by CM Sephadex chromatography. PKC activity was increased 10-fold in the presence of calcium and phosphatidylserine, but no activation by Mg+2-ATP preincubation or inhibition by NaF was observed. In contrast, H4PK activity was increased 8-fold by preincubation with Mg+2ATP and NaF completely inhibited this enzyme. Activators and inhibitors of PKC did not affect H4PK activity. The substrate specificity of the H4PK and PKC also differed substantially. On the basis of these data it is concluded that PKC and H4PK are not related enzymes.