The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.