Although many lines of evidence indicate that the cellular protein p107 is closely related to the retinoblastoma protein, the exact function of the p107 gene and its regulation are presently not known. To investigate the molecular mechanism controlling expression of the human p107 gene, a 5' flanking sequence of this gene was isolated and shown to promote high-level expression of a luciferase reporter gene in cycling human 293 and Saos-2 cells. Sequencing and transcription mapping analyses showed that the human p107 promoter is TATA-less and contains a tandem, direct repeat of E2F-binding sites, with the 3' copy overlapping the major transcription initiation site. Deletion analysis of the p107 promoter showed that a promoter DNA fragment containing only the two E2F sites together with the leader sequence could direct relatively efficient expression in 293 cells. Site-directed mutagenesis of these E2F sites revealed that although both sites were important for p107 promoter activity, mutation on the proximal, initiation site copy of the E2F site showed a stronger effect. The human p107 promoter could be repressed by the retinoblastoma protein and its own gene product. Interestingly, the repression was found to be mediated through the 5' copy of the E2F site. These studies demonstrate for the first time differential roles of two tandem E2F sites in promoter regulation.