Tryptic peptide analyses were performed on the major structural 52,000- and 36,000-dalton glycoproteins (gp52 and gp36-38) and the nonglycosylated 28,000-, 14,000-, and 10,000-dalton proteins (p28, p14, and p10) of the highly oncogenic murine mammary tumor viruses (MMTVs) of C3H, RIII, and GR mice, i.e., MMTV(C3H), MMTV(RIII), and MMTV(GR), respectively. Each virus was grown in both murine and feline cells to ensure the virus-coded nature of each peptide analyzed. The gp36-38 peptide maps of all three MMTVs were indistinguishable, as were the p14 maps of the different MMTVs. Both the p28 and the gp52 of MMTV(C3H), however, could be clearly distinguished from the corresponding proteins of MMTV(RIII) and MMTV(GR), regardless of whether the viruses were grown in feline or murine cells. The p1o of MMTV(RIII) was clearly different from that of MMTV(C3H) and MMTV(GR). Therefore, tryptic peptide analysis of three proteins, gp52, p28, and p10, can serve to distinguish these three viruses from one another. These studies further characterize the heterogeneity in polypeptides among MMTVs.