A procedure was developed for the quantitation of the major folacin compounds present in foods and other biological materials. Extraction conditions were selected to provide conversion of 10-formyltetrahydrofolic acid (10-HCO-H4folic acid) to 5-HCO-H4folic acid. Polyglutamyl folates were deconjugated with hog kidney conjugase. The resulting folacin monoglutamates were separated by reverse-phase high performance liquid chromatography after extract purification on an anion exchange column. Detection of H4folic acid and its substituted derivatives was performed by monitoring the native fluorescence of the reduced folates in the acidic mobile phase. Folic acid, H2folic acid and also H4folic acid were measured fluorometrically by using an oxidative postcolumn derivatization system in series with the first fluorometer. Detection limits ranged from 0.03 to 2.3 pmol/100 microliters injection for the various folates. The validity of the method was supported by recovery and fluorescence spectral studies and by comparison with Lactobacillus casei assays for a variety of samples. Analysis of rat liver following a single pulse dose of 3H-labeled folic acid indicated large differences between the patterns of radiolabeled and endogenous folates.