Proteins solubilized from normal BALB/3T3 cells and BALB/3T3 transformed by simian virus 40 or Kirsten sarcoma virus have been analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. A large fraction of the polypeptides of the virus-transformed cells, about 30%, were different from normal cells. In contrast to the marked differences between normal and transformed cells, the polypeptides of the DNA and RNA virus-transformed cells were almost identical. These findings were observed with polypeptides stained by Coomassie Blue, or labeled with [14C]glucosamine or [35S]methionine. Pulse-chase analysis showed that most of the polypeptides were stable during 20 hr of incubation. The identity of several polypeptides was confirmed by tryptic peptide mapping.