The regulation of non-autonomous retrotransposition is not known. A recombinant bearing a hygromycin gene and a viral-like 30 (VL30) retrotransposon tagged with an enhanced green fluorescent protein (EGFP) gene-based retrotransposition cassette was constructed and used for detection of retrotransposition events. Transfection of this recombinant produced retrotransposition events, detected both by EGFP fluorescence and PCR analysis, in hygromycin-selected clones of two established simian virus 40 (SV40)-transformed mouse NIH3T3 cell lines but not in normal NIH3T3 cells. The retrotransposition potential of this recombinant, as a provirus, was studied in stably transfected NIH3T3 clones. Transfection of these clones with either a wild-type or a mutant LE1135T SV40 large T antigen gene, not expressing small t protein, induced retrotransposition events at high frequencies as measured by fluorescence-activated cell scanning (FACS). In addition, measuring retrotransposition frequencies over a period of nine days following infection with isolated SV40 particles, revealed that the frequency of retrotransposition was time-dependent and induced as early as 24 h, increasing exponentially to high levels (>10(-2) events per cell per generation) up to nine days post-infection. Furthermore, ectopic expression of a cloned MoMLV-reverse transcriptase gene also produced retrotransposition events and suggested that the large T antigen most likely acted through induction of expression of endogenous reverse transcriptase genes. Our results show a direct correlation between SV40-cell transformation and VL30 retrotransposition and provide for the first time strong evidence that SV40 large T antigen up-regulates the retrotransposition of VL30 elements.