Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.