Various polyomavirus-transformed hamster cell lines derived from tumors or from infected hamster cell cultures synthesized polyoma middle and small tumor (T)-antigens but no full-size large T-antigen. Instead, all cell lines produced the same or similar polyoma T-antigen-related proteins of ca. 61 kilodaltons (kDal). Like large T-antigen synthesized in lytically infected mouse cells, the 61-kDal proteins were phosphoproteins showing electrophoretic and charge heterogeneities. Chromatographic analysis of the methionine-containing tryptic peptides indicated that the 61-kDal proteins were truncated forms of large T-antigen comprising amino acid residues 1 to 485 (+/- 25). Analysis of viral DNA present in hamster chromosomal DNA of three independently isolated cell lines confirmed that synthesis of the 61-kDal proteins was due to a discontinuity in the large T-antigen coding sequence, most likely located between 7 and 8.9 map units on the polyoma DNA map. The three cell lines yielded essentially the same patterns of viral DNA-containing restriction enzyme fragments, suggesting that insertion of viral DNA into the hamster chromosomes took place at closely similar sites.