Production of fumonisins by Fusarium moniliforme on naturally infected maize ears is an important food safety concern due to the toxic nature of this class of mycotoxins. Assessing the potential risk of fumonisin production in developing maize ears prior to harvest requires an understanding of the regulation of toxin biosynthesis during kernel maturation. We investigated the developmental-stage-dependent relationship between maize kernels and fumonisin B₁ production by using kernels collected at the blister (R2), milk (R3), dough (R4), and dent (R5) stages following inoculation in culture at their respective field moisture contents with F. moniliforme. Highly significant differences (P  0.001) in fumonisin B₁ production were found among kernels at the different developmental stages. The highest levels of fumonisin B₁ were produced on the dent stage kernels, and the lowest levels were produced on the blister stage kernels. The differences in fumonisin B₁ production among kernels at the different developmental stages remained significant (P  0.001) when the moisture contents of the kernels were adjusted to the same level prior to inoculation. We concluded that toxin production is affected by substrate composition as well as by moisture content. Our study also demonstrated that fumonisin B₁ biosynthesis on maize kernels is influenced by factors which vary with the developmental age of the tissue. The risk of fumonisin contamination may begin early in maize ear development and increases as the kernels reach physiological maturity.