Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.