Expression of the polyketide synthase (pks) gene which is involved in ochratoxin A (OTA) biosynthesis in Aspergillus ochraceus is linked to production of the mycotoxin, with high levels of pks mRNA accumulation occurring in cultures producing OTA, as assessed by real-time reverse transcription (RT)-PCR. OTA production is regulated by nutrient availability, with supplementation of OTA restrictive potato dextrose broth with yeast extract resulting in a 39-fold increase in production of the mycotoxin. This effect appears to be mediated at the level of gene transcription as there is a concomitant increase in pks mRNA accumulation. OTA production is also strongly influenced by culture pH with large amounts of OTA being produced at pH values <7.0 with reduced amounts being produced at higher pH values. pks transcript levels again mirrored the OTA production profile observed at the different pH values. The transcription of two putative p450 type monooxygenase genes, namely p450-H11 and p450-B03 genes closely mirrored that of the pks gene under all growth conditions tested, suggesting their involvement together with the pks in OTA biosynthesis. The expression profile of the p450-B03 gene in particular is very similar to that of the pks gene, indicating that this gene may be clustered with the pks as part of the OTA biosynthetic gene cluster.