Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in fumonisin C-10 hydroxylation or that do not produce any fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.