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Infection of chicken embryo fibroblasts by Rous sarcoma virus induces a variety of alterations in cellular growth and morphology. We have used two-dimensional polyacrylamide gel electrophoresis to examine the effects of viral transformation on the pattern of synthesis and phosphorylation of cellular polypeptides. Infection by Rous sarcoma virus does not appear to induce the de novo synthesis, or the complete Transformation of fibroblasts by DNA or RNA tumor viruses results in many changes in cellular morphology and metabolism (1). In Rous sarcoma virus (RSV), these changes occur in response to the expression of a single viral gene, src. The product of this gene has recently been identified as a phosphorylated polypeptide, Mr 60,000, denoted pp6Osrc (2-4). The finding that pp60src is associated with protein kinase activity (3,4) suggests that some or all of the effects of the src gene may be mediated by the phosphorylation of cellular polypeptides. Several proteins, such as fibronectin, collagen, glucose transport proteins, and hyaluronic acid synthetase, are present in altered quantities in transformed cells (1). This suggests that the initial phosphorylation event leads to an altered pattern of polypeptide synthesis in the transformed cell.To investigate the effects of transformation on the synthesis and phosphorylation of cellular polypeptides, we have used two-dimensional polyacrylamide gel electrophoresis to examine the radiolabeled polypeptides of chicken embryo fibroblasts infected either with nondefective (nd) RSV or with transformation-defective (td) or temperature-sensitive (ts) mutants. Our (Amersham, Ci/mmol; 1 Ci = 3.7 X 1010 becquerels). Cells to be labeled with [32P]orthophosphate were labeled in phosphate-free medium supplemented with 4% calf serum, 1% chicken serum, and 2.5 mCi of [32P]orthophosphate per ml (ICN, carrier-free).Cell Lysis.[35S]Methionine-labeled cultures were washed twice with phosphate-buffered saline and the cells were lysed directly in the oulture wells at 0.5 mg of protein per ml of lysis buffer (10). The contents were scraped with a Teflon scraper,