We describe here cloning and characterization of the promoter region of the human ribonucleotide reductase R2 gene. Primer extension and sequence data indicated that two different transcripts were produced via using two different promoter regions, Promoter activity of the 5' flanking region of the first transcript was approximately 100-fold higher than controls, and that of the second transcript was approximately 30-fold higher than controls, Particularly, the proximal region of the first transcript, -125 to +1 bp, was responsible for approximately a 50-fold increase in promoter activity, compared to controls. This region had three CCAAT sequences, each of which contributed similarly to promoter activity. When all three CCAAT sequences were mutated, promoter activity declined 80%. In addition, the promoter region -125 to fl bp was responsible for cell-cycle-specific expression. These data provided essential information concerning regulatory mechanisms of cell-cycle-specific expression of human ribonucleotide reductase R2.