Microsatellite instability (MSI) has been detected in endometrial carcinomas occurring in women affected by hereditary nonpolyposis colorectal carcinoma (HNPCC) as well as in 20% of presumably sporadic endometrial tumors. While the MSI+ phenotype observed in endometrial tumors from HNPCC patients is attributed to germ line mutations in mismatch repair (MMR) genes, somatic mutations of known MMR genes are infrequent in MSI+ sporadic endometrial carcinomas. Recently, cytosine methylation of the MLH1 promoter region has been identified in a subset of MSI+ colon primary carcinomas and cell lines. We studied the MLH1 and MSH2 promoter methylation status in 29 presumably sporadic uterine endometrioid carcinomas (UECs), which had previously been characterized for the MSI phenotype and a subset for DNA MMR gene mutational status. We found that 13 (45%) of 29 cases of EC were hypermethylated in the 5' CpG island of MLH1. Hypermethylation of MSH2 was not observed. MLH1 was hypermethylated in 12 (92%) of 13 MSI+ tumors, while only 1 (6%) of 16 MSI- tumors (Fischer's exact test P<O.0001). Other tumor types we tested did not demonstrate MLH1 promoter hypermethylation. Our data suggest that hypermethylation of MLH1, but not of MSH2, is associated with the MSI phenotype in sporadic endometrial carcinomas.