Plasma pharmacokinetics of high-dose (500 mg/m2) leucovorin calcium (dl-5-formyltetrahydrofolic acid [dl-CF]) and fluorouracil (FUra) have been evaluated in patients with advanced colorectal cancer treated with the combination of FUra and dl-CF by two different intravenous (IV) schedules: (A) In patients with no prior chemotherapy, dl-CF was administered by a two-hour IV infusion and FUra by rapid IV injection one hour after the start of the dl-CF infusion and (B) in previously treated patients, dl-CF and FUra were administered by five-day continuous IV infusion (CI). Following the two-hour infusion of dl-CF, mean peak plasma concentration and elimination half-life of I-5-formyltetrahydrofolic acid (I-CF) were 24 +/- 6 mumol/L and 0.8 +/- 0.1 hour, respectively. CI of dl-CF over five days yielded a mean steady-state plasma level of I-CF of only 1.2 +/- 0.5 mumol/L. Peak and steady-state plasma concentrations of the metabolite 5-methyl tetrahydrofolic acid were comparable in the two schedules (17 +/- 8 mumol/L for the two-hour infusion and 12 +/- 5 mumol/L for the CI). Areas under the concentration v time curve (AUC) of total reduced folates were significantly greater under conditions of CI: 89.0 v 16.7 mmol/L/min for the two-hour infusion. In tumor tissue, 5,10-methylenetetrahydrofolate increased eight-fold two to four hours following the two-hour infusion and two-fold during the CI of dl-CF and FUra. Inhibition of thymidylate synthase (dTMP-S) by the two-hour and CI infusion schedules were 66% v 39%, respectively. The observed differences in the intracellular dTMP-S folate cofactor pools and the degree of inhibition of dTMP-S achieved in patients treated by two different schedules may be due to differences in the biochemical properties and/or to differences in the modulation of FUra metabolism by folate of tumor tissues obtained from newly diagnosed and previously treated patients.