Fumonisins are polyketide mycotoxins produced by the maize pathogen Fusarium verticillioides and are associated with multiple human and animal diseases. A fumonisin biosynthetic pathway has been proposed, but structures of early pathway intermediates have not been demonstrated. The F. verticillioides FUM6 gene is required for an early pathway step. Here, metabolites produced by strains of the fungus with an inactivated FUM6 gene were purified and shown by mass spectrometry and NMR spectroscopy to have fumonisin-like structures but without substitutions at C-14 and C-15. The major metabolite was 2-amino-12,16-dimethylicosane-3,10-diol. Lesser amounts of 3-keto and triol analogues of the metabolite were also identified. In precursor feeding experiments, 2-amino-12,16-dimethylicosane-3,10-diol was transformed to fumonisins by a F. verticillioides strain with an inactive fumonisin polyketide synthase gene. The results support the hypothesis that the FUM6-encoded enzyme catalyzes fumonisin C-14 and C-15 hydroxylation and provide direct spectroscopic and biochemical evidence for structures of early intermediates in fumonisin biosynthesis.