Tumors with a defective DNA mismatch repair system (MSI-H tumors) have distinct molecular and clinicopathologic profiles compared with mismatch repair-proficient tumors and are associated with a relatively favorable prognosis. There is evidence to suggest that colorectal cancer patients with MSI-H tumors respond differently to adjuvant chemotherapy. Determination of MSI status also has clinical application for assisting in the diagnosis of suspected hereditary nonpolyposis colorectal cancer cases. For these reasons, it is becoming increasingly apparent that testing for MSI should be conducted routinely in human cancer types that frequently present with such a phenotype. BAT-26 and BAT-25 are mononucleotide repeats that are widely used to establish the MSI status of human tumors. We show here that their allelic size profiles provide an estimate of the percentage of contaminating normal cells in MSI-H tumors. These markers are sensitive enough to detect instability when the tumor cell content of a sample is as low as 5-10%. MSI-H tumors contain mutations in coding repeats within genes known to be targets for instability. In cases with low tumor cell content, no mutations in any of 9 coding repeats were detected. However, when these samples were enriched for tumor cells, mutations were detected in the same target genes. Thus, BAT-26 and BAT-25 markers accurately identify MSI-H tumors without prior need for enrichment for tumor cells and indicate which samples require further purification before screening for mutations in target genes for instability. Our results have implications for large-scale screening of cancer patients to determine MSI-H status and prognosis.