Irinotecan has shown activity in advanced colorectal cancer resistant to leucovorin and fluorouracil. Preclinical experiments on cell cultures and human tumor xenografts indicated potential synergy when combining irinotecan and fluorouracil. We designed a new regimen combining leucovorin, fluorouracil, irinotecan, and hydroxyurea (FOLFIRI-2) and conducted a phase II study to establish its efficacy and tolerance in advanced colorectal cancer refractory to fluorouracil and oxaliplatin. Treatment was repeated every 2 weeks and consisted of leucovorin 400 mg/m2 on day 1, immediately followed by 46 hours of continuous infusion of fluorouracil 2,000 mg/m2, irinotecan 180 mg/m2 on day 3, and hydroxyurea 1,500 mg the day before leucovorin, and on days 1 and 2. Treatment was continued until progression or limiting toxicity. Twenty-nine heavily pretreated patients entered the study. Five patients achieved an objective response (17%), and 12 obtained stabilization of disease or minor response (52%). Five patients failed to continue treatment (17%) because of toxicity or worsening condition. From the start of FOLFIRI-2 treatment, median progression-free survival was 4.1 months and median survival was 9.7 months. Grade III/IV National Cancer Institute-Common Toxicity Criteria toxicities were nausea 17%, diarrhea 31%, mucositis 14%, neutropenia 52%, and febrile neutropenia 14%. FOLFIRI-2 achieved a good rate of response and stabilization in heavily pretreated patients despite significant toxicity.