High-performance liquid chromatography (HPLC) was investigated as a technique for determination of moniliformin, a toxic secondary metabolite of various Fusarium species. Two HPLC procedures gave satisfactory results. In the first procedure, separation was achieved on a strong anion exchange column (10 microns, 4 mm id x 25 cm) with an eluting solvent consisting of 0.01 M sodium dihydrogen phosphate (pH 5.0) and a flow rate of 1.0 mL/min. The second procedure made use of paired ion chromatography on a reverse phase column (10 microns, 4 mm id x 25 cm). Elution was done at 2 mL/min with 0.005 M tetrabutylammonium hydrogen sulphate in a mixture containing 8% methanol and 92% 0.1 M sodium phosphate buffer (pH 7.0). Moniliformin was detected in both procedures by ultraviolet absorbance at 229 nm. The lower detection limit of pure moniliformin was 1 ng per injection. Water extraction of moniliformin from dried Fusarium cultures grown on maize was found to be efficient, giving a 95% recovery from a spiked sample. Peak height and retention time reproducibility was good for both procedures. In the analysis of maize containing on the order of 1 mg/kg moniliformin, background interference made interpretation of chromatograms difficult. The cleanup procedure giving the most promising results is described. HPLC methods have been applied successfully in screening fungal cultures for moniliformin production and for monitoring the steps in isolating moniliformin from mold material.