We present a rapid, sensitive, and automated HPLC method with direct resolution of l-folinic acid (l-FA), d-folinic acid (d-FA), and 5-methyltetrahydrofolate (5MTHF) from plasma samples. Plasma (500 microL) is first extracted on solid phase (RP-18 cartridge). The dI-FA peak is collected on-line from a reversed-phase column (RP-8, 119 x 2 mm, 4 microns: HPLC 1) and then automatically loaded onto a chiral stationary phase (human serum albumin, 150 x 4.6 mm, 7 microns: HPLC 2). The same mobile phase flows in both systems (0.2 mol/L Na2HPO4:1-propanol, 98:2, pH 6.2). HPLC 1 allows quantification of 5MTHF by absorption at 313 nm; HPLC 2, the quantification of l-FA and d-FA by electrochemical detection in the oxidation mode (total run time 18 min). Recoveries are > 80%. CVs for intra- and interassay reproducibilities are < 5% and 15%, respectively. Linearity of the response (0.1-1 mumol/L and 1-50 mumol/L, r = 0.99, P < 0.01) is satisfactory. The sensitivity limit is 50 nmol/L for 5MTHF and 20 nmol/L for l-FA and d-FA. This assay is substantially improved over existing methods regarding feasibility and is being used in pharmacokinetic investigations in cancer patients.