In vitro studies suggest that a deficient mismatch repair (MMR) system reduces 5‐Fluorouracil cytotoxicity. Colon cancer (CC) in hereditary nonpolyposis colorectal cancer (HNPCC) is due to a dysfunctioning MMR gene that leads to microsatellite instability (MSI). Clinical studies on the efficacy of 5‐Fluorouracil (5‐FU) in MSI high tumours are contradictory. In a retrospective study, we compared the survival of subjects with stage III CC from HNPCC families that were treated with and without adjuvant 5‐FU. The Dutch HNPCC family registry was used. Information on adjuvant chemotherapy for stage III CC was obtained from subjects of families with a mutation and/or who fulfilled the AMS criteria or who were strongly suspicious for HNPCC. CC specific survival was calculated. Observation time was measured either until the date of death, date of a second primary CC or until the closing date of the study, i.e., June 1, 2001. Statistical analysis was done by Kaplan‐Meier survival analysis. A total of 92 subjects with stage III CC were included. Twenty‐eight of them (17 males) had adjuvant treatment with 5‐FU. The median follow‐up was 4 (range: 1–17) years; 8 subjects died of CC. The 5‐year survival was 70% (95% Cl: 49–90). Sixty‐four subjects (36 males) did not have adjuvant therapy. Their median follow‐up was 6 (range: 0–23) years. Twenty of them died of CC. The 5‐year survival in this group was also 70% (95% Cl: 59–83). To date, the selection of patients with CC for 5‐FU treatment is based on the stage rather than the biology of the tumour. In our study, the 5‐year survival of subjects treated with and without adjuvant 5‐FU did not differ. Further studies are necessary to elucidate the role of MSI in 5‐FU treatment of MSI‐H tumours in HNPCC. © 2004 Wiley‐Liss, Inc.