Human adenovirus type 9 (group D) induces mammary fibroadenomas in female rats at a high frequency. Ad9 has been used to transform an established rat embryo fibroblast cell line, 3Y1. These cells were transformed very efficiently by Ad9 and the frequency of transformation was about 16 times higher than that of adenovirus type 2 (group C). Three transformed cell lines (92.2, 93.1, and 96.2) were analyzed for the content and expression of viral DNA sequences. The Ad9-transformed cell lines contained about 7 to 30 copies of integrated viral DNA. It was found that all or almost all of the viral genome was integrated in a linear form and probably exists as tandem repeats in a head-to-tail orientation. In this respect, Ad9-transformed cells resemble Ad12-transformed cells which also contain integrated nearly complete copies of the viral genome. The Ad9-transformed cells have been shown to express the E1A and E1B regions at the RNA level.