It is important to know the regulation of the expression of the mac25 gene because of its reduced expression in several cancer cells and of its induction by some hormonal factors. We cloned the promoter region of the murine mac25 gene and found five repeats of CCAAT sequences, four Sp1 sites, a TATA-like sequence, and an initiator (INR) sequence. Analysis using luciferase reporter plasmids indicated that CCAAT repeats have a strong enhancer activity and the second to fourth Sp1 sites are essential for basal activity of the expression of the mac25 gene. The 1 kb region that contains the promoter and exon 1 of the mac25 gene was in a typical CpG island. As hypermethylation and reduced expression of the mac25 gene were reported in murine liver tumors, methylation of this CpG island may be directly associated with the expression of the mac25 gene and tumorigenesis.