Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (gamma 32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by trypsin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphorylated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein phosphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.