Research on aflatoxin (AF) production has traditionally focused on defining the AF biosynthetic pathway with the goal of identifying potential targets for intervention. To understand the effect of nitrogen source, carbon source, temperature, and pH on the regulation of AF biosynthesis, a targeted cDNA microarray consisting of genes associated with AF production over time was employed. Expression profiles for genes involved in AF biosynthesis grouped into five clades. A putative regulon was identified consisting of 20 genes that were induced in the conducive nitrogen and pH treatments and the non-conducive carbon and temperature treatments, as well as four other putative regulons corresponding to each of the four variables studied. Seventeen genes exhibited consistent induction/repression profiles across all the experiments. One of these genes was consistently downregulated with AF production. Overexpression of this gene resulted in repression of AF biosynthesis. The cellular function of this gene is currently unresolved.