Leucovorin (LV) is commonly used as a modulator of 5-fluorouracil (5-FU) cytotoxicity. In patient with colon cancer, the addition of LV to 5-FU improves response rates, and in some trials has improved survival in advanced disease and in the adjuvant setting. Leucovorin is generally administered as a racemic mixture, but the isomers differ substantially in pharmacokinetics and biological activity, with 6S-LV the predominant active component. The current study was undertaken to determine the effect of 6R on the pharmacokinetics of 6S-LV, and to characterize the toxicity and antitumor effect of 5-FU when administered with 6S-LV to patients with advanced colorectal carcinoma. Thirty patients were treated with weekly 5-FU plus high dose 6S-LV. To determine the effects of 6R-LV on the pharmacokinetics of S6-LV, 20 patients were randomly assigned to receive either 250 mg/m2 6S-LV or 500 mg/m2 6R,S-LV as a 2 hour IV infusion on day -2, and the other preparation on day -1, with pharmacokinetics measured each day. The presence of 6R-LV had no effect on the AUC, Clp, Cmax, or terminal phase t1/2 of 6S-LV. The overall response rate was 40% (C.I. 23-60%). The most frequent toxicities were gastrointestinal. In this small cohort, scheduled and delivered dose intensity was positively associated with response (p = 0.05). These results show that there is no pharmacokinetic advantage to the use of 6S-LV rather than 6R,S-LV as a modulator of 5-FU.