Mismatch repair (MMR) deficiency in tumours from patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is mainly caused by mutations in the MLH1, MSH2, and MSH6 genes. A major challenge in the clinical management of patients with suspected HNPCC is the frequent occurrence of missense mutations in MSH6. These can be considered neither deleterious nor clinically innocent a priori. To assess their significance we studied five novel MSH6 missense mutations in six patients derived from a series of consecutive endometrial and colorectal cancer patients selected for study after their tumours were determined to be microsatellite unstable. We tested each mutated protein for heterodimerisation with MSH2 and for in vitro MMR capability. Four mutations (R128L, P623L, K728T, G881K+S) showed no impairment of these functions while the fifth (E1193K) displayed marked impairment of both functions. These results, taken together with our previous similar findings concerning six other missense mutations in MSH6, allow us to conclude that many or most missense changes in MSH6 likely are clinically innocent, whereas some missense changes such as E1193K, which lead to impaired MMR, are likely to be clinically significant, but have low penetrance.