Expression of the Simian virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. Our results demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids lacking the SV40 origin of DNA replication were as proficient as wild type plasmids, indicating that viral DNA replication is not required for cytogenetic damage. We have also shown that chromosome aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. Our results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.