Germline mutations in human mismatch repair genes (mostly in hMSH2 and hMLH1, and recently also in hMSH6) have been found to be associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. HNPCC tumors are phenotypically characterized by a high level of microsatellite instability (MSI-H). Moreover, about 10–15% of sporadic colorectal cancers are MSI-H, predominantly on the basis of epigenetic silencing of hMLH1 by promoter methylation, whereas somatic inactivation of hMSH2 has rarely been found. In contrast, little is known about the overall involvement of hMSH6 in colorectal cancer. We investigated a series of 212 colorectal cancer specimens, comprising 141 sporadic cases and 71 cases fulfilling Bethesda guidelines for HNPCC for microsatellite instability, for protein expression of the four mismatch repair genes hMSH6, hMSH2, hMLH1 and hPMS2 by immunohistochemistry and for mutations by sequencing. We found different frequencies of abnormal gene expression for each mismatch repair protein studied. Among cases not fulfilling Bethesda guidelines, we identified hMLH1- and hMSH6-deficient cases. Sequence analysis identified hMSH6 germline mutations for almost all hMSH6-deficient cases. Lost expression of one or two of the four proteins was always associated with MSI-H tumors. Conversely, all except one of the MSI-H cases demonstrated lost or aberrant expression of one or more of the proteins, leaving little room for additional genes associated with the MSI-H phenotype. The combination of analysis of microsatellite instability and expression of the four mismatch repair proteins was highly predictive for the respective genes involved.