As part of a clinical trial of cisplatin, 5-fluorouracil (5-FU), and leucovorin (LV) for treatment of patients with advanced head and neck cancer, patients received 100 mg of LV (d,I-5-formyltetrahydrofolate) orally every 4 hours for 5 days. On days 2 and 4 of treatment, plasma samples were obtained 2 hours after (peak) and 30 minutes before (trough) a dose of LV. Total LV and 5-methyltetrahydrofolate (THF) concentrations were measured with high-performance liquid chromatography (HPLC) analysis. LV stereoisomer concentrations were determined by chiral HPLC on a bovine serum albumin-bonded silica column. Thus far, plasma folate levels have been analyzed for ten cycles of treatment administered to 7 patients (40 samples). Administration of LV in divided oral doses approximates a plasma steady state with no significant differences noted between peak and trough concentrations. Mean (fSD) plasma concentrations for all samples were (pmol): LV, 3.2 & 1.3; I-LV, 0.28 f 0.21; CLV, 2.9 & 1.2; and THF, 4.25 f 2.5. Plasma levels of C L V and THF tended to be approximately 10% higher on day 4 than day 2, although mean differences were not significantly different due to substantial interpatient variability. Of note was that the sum of THF and I-LV exceeds that of (I-LV which was consistent with selective absorption of the I-isomer of LV. Mean ratios of CLV/I-LV and CLV/I-LV and THF were 13.7 f 10 and 0.88 f 0.68, respectively. The authors conclude that oral administration of LV in divided dose (1) simulates a continuous intravenous infusion; (2) produces plasma levels of I-reduced folates in a range known to potentiate 5-FU cytotoxicity; and (3) results in low ratios of d/I-reduced folates that may be important in maximizing the effectiveness of 5-FU-LV chemotherapy.