1. The pharmacokinetics and toxicity of racemic 5-methyltetrahydrofolic (rac-5-MTHF) acid after i.v. infusion were investigated in 18 patients with advanced colorectal cancer. Doses of 100-600 mg rac-5-MTHF/m2 were administered over 2 h together with a bolus of 500 mg/m2 5-fluorouracil (5-FU) as a midpoint injection. 2. The pharmacokinetics of both diastereoisomers were linear in the range from 100-600 mg 5-MTFH/m2. Independent of the administered dose, the maximal plasma concentration of [R]-5-MTHF was nearly twice that of [S]-5-MTHF. The elimination of [S]-5-MTHF from plasma was considerably faster than that of the [R]-isomer (elimination half-life: 3.1 +/- 1.0 h vs 8.3 +/- 3.2 h). No metabolites were detected in plasma and in urine samples. 3. The plasma protein binding was stereoselective ([R]-5-MTHF bound: 88.2 +/- 2.7%; [S]-5-MTHF bound: 59.9 +/- 6.8%; P < 0.001), causing a significantly higher renal clearance for [S]-5-MTHF when compared with the [R]-isomer (37.5 +/- 23.7 ml min-1 vs 12.7 +/- 11.2 ml min-1, P < 0.001). There was no dose dependence, but gender influenced renal clearance (CLren[R]-5-MTHF: male vs female: 20.5 +/- 14.5 ml min-1 vs 7.8 +/- 4.7 min-1, P = 0.03; CLren [S]-5-MTHF: male vs female: 57.2 +/- 21.7 ml min-1 vs 25.7 +/- 16.2 ml min-1, P = 0.006). 4. Toxic side effects of the combination 5-FU/5-MTHF were rare and generally mild, and included stomatitis, nausea/emesis, diarrhoea, anaemia, leukopenia, and thrombocytopenia. 5. In combination with 500 mg 5-FU/m2 a single dose of 600 mg rac-5-MTHF/m2 can safely be administered to patients with colorectal cancer. A similar therapeutic benefit of 5-MTHF to folinic acid in the biochemical modulation of 5-FU is supported by the comparison of in vitro and in vivo data.