Although C-MYC is overexpressed in a number of tumors, the mechanisms governing its expression in normal or tumor cells are not completely understood. Recruitment of the Retinoblastoma protein family members to gene promoters by E2F factors has a dominant negative effect on their activity during the G(0) and G(1) phases of the cell cycle. Despite the presence of an E2F-binding site on the C-MYC promoter, it escapes the repressive effect of E2F-Retinoblastoma complexes through unknown mechanisms during exit from quiescence. We hypothesized that occupancy of E2F elements by factors distinct from E2F might account for this escape. To test this hypothesis, we investigated whether the E2F element in the C-MYC promoter is regulated differently than E2F elements in promoters that are activated at the G(1)-S transition. Employing gel shift analysis, the E2F element from the C-MYC promoter was found to form a unique non-E2F complex, referred to as E2F C-MYC Specific (EMYCS), which is not observed with E2F elements from several other promoters. The DNA contact residues for EMYCS are distinct but overlapping with residues required for binding of E2F proteins. Finally, the approximate estimated molecular weight of the DNA-binding component of EMCYS is 105 kDa. Functional studies indicate that EMYCS has transcriptional transactivation capacity and suggest that it is required to activate the C-MYC promoter during exit from quiescence.