Human MLH1 is one of the DNA mismatch repair genes and located in chromosome 3p21.3. Alterations of the MLH1 were associated with various kinds of human tumors. The present study was to investigate allelic loss of MLH1 and microsatellite instability (MSI) in esophageal squamous cell carcinomas (ESCC), and to estimate the correlation between MSI status and allelic loss of the MLH1 gene. The MSI of 14 microsatellite markers and mRNA expression of MLH1 were assessed in a large cohort of patients with ESCC by using denaturing polyacrylamide gel electrophoresis (PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Thirty-five percent of tumors displayed MSI in at least one tested marker. MSI in D3S1611, an intragenic marker of the MLH1, was observed in 66.7% of tumors. No down-expression of MLH1 was found at the transcriptional level. The presence of MSI did not correlate with tumor stage, degree of differentiation, lymphonode-metastasis, age and sex of the patients, neither with allelic loss of the MLH1 gene. The data suggested that LOH of MLH1 is common in ESCC, but not lead to the alteration of MLH1 at the level of RNA expression. MSI in tested microsatellite markers occurs frequently in ESCC but is not associated with allelic loss of the MLH1 gene.