ThehMLH1gene encodes a protein that is involved in the DNA mismatch repair system. The coding region of thehMLH1gene has been known to be mutated in a subset of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Our current research characterized the promoter region of thehMLH1gene and searched for mutations correlating to HNPCC. Utilizing the oligo-capping method, major transcription start sites of thehMLH1gene were mapped at two locations. The core promoter region of about 180 bp was determined by the luciferase assay of serial deletion mutants. Although we did not find any pathogenic mutation in thehMLH1promoter region by PCR-SSCP, we found a single-nucleotide polymorphism at position −93 nt from the adenine residue of the start codon. By PCR-RFLP analysis withPvuII for this polymorphism, we detected LOH in four tumors from three patients. An easy detection of this polymorphism with PCR-RFLP and high incidence (∼50%) of informative cases make this polymorphism a suitable marker for the detection ofhMLH1allelic losses.