The arrangement and localization of viral genomes integrated in RSb cells were analyzed by molecular biological techniques. The RSb cell line was established from human embryonic fibroblasts transformed by sequential infection of Rous sarcoma virus and Simian virus 40 (SV40). SV40 DNA used for analyses was obtained by molecular cloning. The copy numbers of integrated viral genomes were estimated by dot blot hybridization. The arrangement of integrated viral genomes was analyzed by Southern blot hybridization. Chromosomal locations of the genome were studied by in situ molecular hybridization. Most of the integrated viral genomes lack 1.14 kb (about 20% of SV 40 DNA) from the late transcription region which encodes the viral core proteins. The integrated defective 4.1 Kb genome is partially repeated in the host DNA. RSb cells contain the whole large T antigen-coding sequence, and the large T antigen may play a major role in the transformation of this cell line. Results of the dot blot hybridization and in situ hybridization indicate that RSb cells contain about 10 copies of viral genomes per cell and that these genomes are integrated in at least 4 sites of the host chromosomes. These results along with others suggest that there is no unique integration site of SV40 DNA in human cells.