Introduction: Studies with flavopiridol have demonstrated that this agent has in vitro activity in chronic lymphocytic leukemia (CLL) and promotes apoptosis independent of p53 function or prior fludarabine exposure. Based upon this pre-clinical data, a phase I/II study of 24 h flavopiridol was performed. Patients and methods: Patients with previously treated CLL patients were enrolled on two sequentially performed cohorts of 13 patients. Patients in the first cohort received flavopiridol (80 mg/m2 as a 24-h continuous infusion [24 h CI]) every 2 weeks. Patients in the second cohort received flavopiridol (80 mg/m2 as a 24 h CI) for week 1 and then were dose escalated by 20 mg/m2 every 2 weeks to a maximal dose of 140 mg/m2 in the absence of symptoms. Patients received up to 12 doses of therapy. Results: Thirteen patients with fludarabine-refractory or intolerant CLL enrolled in each cohort. Patients received a median of five treatments in each cohort with only two patients completing all 12 courses of therapy. There were no partial or complete responses noted. Toxicity was manageable in most patients and included anemia, thrombocytopenia, infections, diarrhea, and fatigue. Conclusions: Flavopiridol as a 24-h continuous infusion has no clinical activity in relapsed, fludarabine-refractory CLL.