An assay for the separation and quantification of folinic acid in serum was developed using high-performance liquid chromatography with electrochemical detection. Folinic acid was extracted from serum using a C18 minicolumn treated with dibasic ammonium phosphate. The drug was eluted from this column with methanol, which was evaporated under a nitrogen stream at 50 degrees. The mobile phase, pH 3.5 ammonium phosphate buffer-methanol-acetonitrile (93:4:3), was pumped at a flow rate of 3.0 ml/min. The recovery of folinic acid added to human serum was 101.11 +/- 8.5% (mean +/- SD). A plot of folinic acid peak height as a function of concentration was linear over the range of 2.5 X 10(-7) to 2.5 X 10(-6) M. Neither methotrexate nor other reduced folates interfered with the analysis of folinic acid. Sample preparation and analysis can be completed within 2 min of sample collection.