Hereditary nonpolyposis colorectal carcinoma is a major cancer susceptibility syndrome known to be caused by inheritance of mutations in at least four genes such as hMSH2, hMLH1, hPMS1, and hPMS2 which encode components of a DNA mismatch repair system. The hMLH1 genomic locus on chromosome 3p has been cloned and shown to cover ∼58 kilobases of genomic DNA and contain 19 exons. The sequence of all of the intron-exon junctions has been determined and used to develop methods for analyzing each hMLH1 exon for mutations. Using these methods to analyze a 3p-linked hereditary nonpolyposis colorectal carcinoma kindred, we have demonstrated that cancer susceptibility in this family is due to the inheritance of a frame shift mutation in the hMLH1 gene.