aflJ resides within the aflatoxin biosynthetic gene cluster adjacent to the pathway regulatory gene aflR and is involved in aflatoxin production, but its function is unknown. Over-expression of aflJ in the aflatoxin-producing strain 86-10 resulted in increased aflatoxin. In an effort to study the function and regulation of aflJ, strain 649-1 lacking the entire biosynthetic cluster was transformed with either reporter constructs, expression constructs, or cosmid clones and analysed for gene expression or metabolite accumulation. Over-expression of aflJ did not result in elevated transcription of ver-1, omtA or aflR. To determine if over-expression of aflJ leads to an increase in early pathway intermediates, strain 649-1 was transformed with cosmid 5E6 and either gpdA::aflJ alone, gpdA::aflR alone, or aflJ and aflR together. Cosmid 5E6 contains the genes pksA, nor-1, fas-1, and fas-2, which are required for the biosynthesis of the early pathway intermediate averantin. 649-1 transformants containing 5E6 alone produced no detectable averantin. In contrast, 5E6 transformants with gpdA::aflR produced averantin, but only half as much as those transformants containing both aflR and aflJ. Northern blot analysis showed that 5E6 transformants containing both aflR and aflJ had five times more pksA transcripts and four times more nor-1 transcripts than 5E6 transformants containing gpdA::aflR alone. Further, aflJ transcription was regulated by aflR. Over-expression of aflR resulted in elevated aflJ transcription. aflJ appears to modulate the regulation of early genes in aflatoxin biosynthesis.