Expression of the hMLH1 gene, one of the DNA mismatch repair genes, is frequently repressed in various cancers such as colorectal, ovarian, gastric, and endometrial origins with a microsatellite instable phenotype. In this study, we investigated details of the relationship between the transcriptional activity and the protein-binding sites in the 5'-flanking region of the hMLH1 gene. Luciferase reporter gene analysis with a series of deletion mutants revealed that a region containing -301 to -76 relative to a translation start site is essential for maximal expression. Eight protein-binding sites in this region were identified by in vivo methylation footprinting analysis and homology search. A presence of binding proteins to CCAAT-box at -145 to -139 was confirmed by the electrophoresis mobility shift assay. Partial involvement of NF-Y was seen by the super gel shift assay. Three reporter plasmids having a single site-directed mutation at -163 to -158, -145 to -139, or -96 to -93 showed 14-30% less activities to that of having the wild-type. Dual or triple mutations were no greater effect than the single mutation on the activity. These results indicate that three cis-elements are essential for full expression of the hMLH1 gene and may work co-operatively.