Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary colon cancer syndrome and is responsible for as many as 10% of all colorectal cancers. Hereditary nonpolyposis colorectal cancer is autosomally dominant with a prevalence of 1 in 200-2000 and exhibits incomplete penetrance. Affected individuals have an approximately 70% lifetime risk of colon cancer with a mean age of onset of 44 years and an approximately 40% lifetime risk of endometrial cancer. At least 5 mismatch repair genes (MLH1, MSH2, MSH6, PMS1, PMS2) have been implicated in HNPCC; however, no predominant mutations were found in these genes. Mutation detection by direct sequencing has proven to be the most sensitive method. We have developed high-throughput full-length sequencing assays of the MLH1, MSH2, and MSH6 genes. These 3 genes account for approximately 90% of all germline mutations found in HNPCC. In our assays, 19 exons of MLH1, 16 exons of MSH2, 10 exons of MSH6, and the adjacent splice sites were amplified using polymerase chain reaction and loaded onto a capillary sequencing machine. Results were analyzed using sequence analysis software and stored in a relational database. Our assay method was validated using 15 affected patients and normal controls. It is anticipated that our high-throughput assay technique will provide accurate diagnoses for patients at risk for HNPCC and thereby facilitate early curative intervention.