Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Most of the genes involved in the biosynthesis of aflatoxin are contained within a single cluster in the genome of these filamentous fungi. Studies directed toward understanding the molecular biology of aflatoxin biosynthesis have led to a number of important discoveries. A pair of fatty acid synthase genes were identified that are involved uniquely in aflatoxin biosynthesis. Two genes were also characterized that represent new families of cytochrome P450 monooxygenases. Gene expression is coordinated during aflatoxin production and is under the control of a positive regulatory gene belonging to a family of fungal transcriptional activators associated with various metabolic pathways in fungi.