Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [gamma-(32)P]ATP or labeling of intact cells with Na(2)H(32)PO(4). Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell lines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [gamma-(32)P]ATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phosphorylation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity.