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Microsatellite instability (MSI) is the phenotypic molecular characteristic of the majority of tumors associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). Women in this group have an increased risk of endometrial cancer (EC). This study aimed to determine whether MSI could be demonstrated in blind endometrial samples from women with EC, HNPCC kindreds undergoing screening for EC, and women with normal endometrium. Twenty-four women with EC, 20 women from HNPCC kindreds, and 20 women undergoing gynecological surgery for benign indications underwent blind sampling. MSI analysis was performed by conventional polymerase chain reaction using fluorescent-labeled primers and automated analysis. Twelve microsatellites were studied with MSI defined as evident when novel alleles were seen in endometrial biopsy samples compared to genomic DNA. Of the 24 EC samples obtained, sufficient DNA for analysis was extracted in 17 cases. Three cases had evidence of MSI in at least 7/12 loci. None of the endometrium from the two other study groups revealed evidence of MSI. This is the first demonstration of MSI in blind endometrial biopsies. The ability to demonstrate MSI in heterogenous endometrial samples suggests potential for the development of a novel EC screening tool for women in HNPCC kindreds.