A method for the production and purification of radiolabeled fumonisin that involves the addition of 14C-acetate to liquid cultures of Fusarium moniliforme in shake flasks is reported. Stable isotope 13C labeling studies were carried out using specifically enriched acetate and several amino acids to determine the location of labeled carbon atoms in the radiolabeled fumonisin that was also produced (650 μCi/mmol). These experiments determined that the 14C was distributed throughout the molecule making it useful for studies of fumonisin residues in animal products. Additionally, the 13C studies indicated that the biosynthesis of fumonisin involves the addition of methionine, glutarate, and serine or alanine to the hydrocarbon backbone. These data best fit the hypothesis that this backbone is polyketide in origin as opposed to being a modified lipid.