Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec- (for secondary metabolism negative) variants were generated through serial transfer of mycelia from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. Previous studies have shown no difference in the DNA sequence of aflR, a positive regulator of AF production, in the sec+ and sec- strains. In this study, AflJ, another positive regulator of AF production, laeA, a global regulator of secondary metabolism, and the intergenic region between aflR and aflJ, were analysed to determine if they play a role in establishment of the sec- phenotype. The study showed that while this sequence identity extended to the aflJ as well as the aflJ-aflR intergenic region, expression of aflR in the sec- strain was several fold lower than that observed in the sec+ strain, while aflJ expression was barely detectable in the sec- strain. Western blot analysis indicated that despite AflR protein being present in the sec- strain, no toxin production resulted. Introduction of a second copy of aflR into the sec- strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec- strains. These results suggest that although aflR, aflJ and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.