We report a simple method that permits simultaneous detection of multiple protein kinase activities using postnuclear supernatant of v-src transformed NIH3T3 cells. A supernatant is incubated with activators of protein kinases and [gamma-32P]ATP, and the phosphorylated proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The method enables detection of activities of at least four protein kinases (protein kinase A, protein kinase C, protein tyrosine kinase(s), and calmodulin-dependent protein kinase III) on a single gel. Experiments using various specific activators and inhibitors of protein kinases indicated that this method can, in crude preparations, reliably detect the protein kinase activities intended for measurement. Protein kinase C activity disappeared when membranes were solubilized, demonstrating the importance of membrane environment for its function. This method should be particularly useful for evaluating and screening protein kinase inhibitors.