A rat cell line, 3Y1, was transfected with the left-end DNA fragments of adenovirus (Ad) 31 DNA and transformed cell lines were established. A cell line 31BY4-1, which was induced by Ad31 BamHI-B (left-most 17.4% of the genome), showed typical transformation phenotypes, while cell lines 31GY1-2, 31GY2-5, and 31GY3-4, which were induced by Ad31 HindIII-G (left-most 6.7% of the genome), showed intermediate phenotypes between transformed and untransformed cells, with the following properties: (1) percent plating efficiencies in Eagle's minimum essential medium with 2% fetal calf serum and in soft-agar culture were extremely low, like those of 3Y1 cells; (2) they form tumors in newborn rats only after long latent periods. Southern blot hybridization revealed that, in all the transformed cell lines, viral DNA sequences were integrated at multiple loci in large-molecular-weight cell DNAs. Northern blot hybridization showed that viral mRNAs of early region 1A (E1A) and early region 1B (E1B) were both transcribed in 31BY4-1 cells as well as in KB cells early after infection with Ad31. On the other hand, E1B viral mRNA was present in a much lesser amount in 31GY cell lines, or was undetectable.