Background: Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumours. Methods: By thorough molecular and clinical evaluation of 41 families, two dif ferent groups were characterised: group 1, 25 families with truncating mutations in MLH1 or MSH2 (12 novel mutations); and group 2, 16 Amsterdam positive famili es without mutations in these genes and without microsatellite instability in th eir corresponding tumours. Results: Significant clinical differences between the se two groups were found. Firstly, earlier age of onset for all colorectal cancers (median 41 v 55 years; p < 0.001) and all tumours (median 4 3 v 56 years; p = 0.022) was observed, comparing groups 1 and 2. Secondly, 68%o f the index colorectal cancers were localised proximally of the splenic flexure in group 1 compared with 14%in group 2 (p < 0.010). Thirdly, more synchronous a nd metachronous colorectal (p = 0.017) and extracolorectal tumours (p < 0.001) w ere found in group 1. Fourthly, a higher colorectal adenoma/carcinoma ratio (p = 0.030) and a tendency towards more synchronousor metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carc inomas. As three mutation negative tumours revealed chromosomal instability afte r comparative genomic hybridisation, these tumours may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway. Concl usion: These data show that HNPCC includes at least two entities with clinical a nd molecular differences. This will have implications for surveillance programme s and for cancer research. Background: Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumors. Methods: By thorough molecular and clinical evaluation of 41 families, two dif ferent groups were characterized: group 1, 25 families with and group 2, 16 Amsterdam positive famili es without mutations in these genes and without microsatellite instability in th eir corresponding tumours. Results: Significant clinical differences between the se two groups were found. , earlier age of onset for all colorectal cancers (median 41 v55 years; p <0.001) and all tumors (median 4 3 v 56 years; p = 0.022) were observed, comparing groups 1 and 2. Secondly, 68% of the index colorectal cancers were localized proximally of the splenic flexure in group 1 compared with 14% in group 2 (p <0.010). Thirdly, more synchronous a nd metachronous colorectal (p = 0.017) and extracolorect al tumours (p <0.001) w ere found in group 1. Fourthly, a higher colorectal adenoma / carcinoma ratio (p = 0.030) and a tendency towards more synchronousor metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carc inomas. As three mutation negative tumors revealed chromosomal instability afte r comparative genomic hybridisation, these tumors may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway. Concl usion: These data show that HNPCC includes at This will have implications for surveillance programs and for cancer research.