Abstract Background These analyses compare the safety and efficacy of 2 forms (levogyre [L] and dextro-levogyre [DL]) of leucovorin (LV) when used with 5-fluorouracil (5-FU) for the adjuvant treatment of patients with stage II and III colon cancer. Materials and Methods The analysis used primary efficacy and safety data of a phase III trial comparing monthly 5-FU/LV or bimonthly LV5FU2 (LV 200 mg/m 2 intravenously over 2 hours followed by 5-FU 400 mg/m 2 bolus and then 600 mg/m 2 continuous intravenous infusion over 22 hours, days 1 and 2, every 2 weeks). In both regimens, depending on the choice made by each center, patients received either DL-LV (200 mg/m 2) or L-LV (100 mg/m 2). Results L-LV and DL-LV were administered respectively to 60% (n = 519) and 40% (n = 357) of the patients. Important prognostic characteristics were well balanced between the 2 groups. The proportion of any grade 3/4 toxicity was 20% in the L-LV group and 17% in the DL-LV group. There was no statistical difference in terms of toxicity between the 2 groups. The median follow-up time was 6.1 years. There were no statistically significant differences between L-LV and DL-LV in terms of either disease-free survival (66.7% vs. 67.2%; hazard ratio [HR], 1.03; 95% CI, 0.82-1.31; P = .78) or overall survival (78.2% vs. 74.5%; HR, 1.28; 95% CI, 0.97-1.69; P = .078). Conclusion This study supports the use of either DL (200 mg/m 2) or L (100 mg/m 2) LV in association with 5-FU as adjuvant treatment of patients with colon cancer.