The ochratoxin A (OTA) polyketide synthase otapks gene has been cloned from Penicillium verrucosum. A P. verrucosum mutant in which the otapksPV gene has been interrupted cannot synthesize ochratoxin A. The protein is most similar to the citrinin polyketide synthase CtnpksMa from Monascus anka (83% identity at the amino acid level). Different nutritional conditions influence OTA production in P. verrucosum, with the addition of glycerol and galactose to MCB resulting in approximately 19 and 32 fold increases in OTA production respectively. These effects are mirrored in increased levels of otapksPV gene transcription. In contrast, the addition of glucose to MCB containing galactose results in an approximate 10 fold repression in OTA production, with this repression again being mirrored in decreased levels of otapksPV gene transcription. Thus the effects of different carbon sources on OTA production in P. verucosum appear to be regulated at the level of gene transcription. Two additional open reading frames, otaE and otaT, were identified in the 5' and 3' flanking regions of otapksPV, respectively. The otaT and otaE genes are co-expressed with P. verrucosum otapksPv, indicating a possible role for these genes in OTA biosynthesis. Furthermore, otaT and otaE were identified as putative homologues of the M. anka citrinin transporter ctnC (72% amino acid identity) and M. anka citrinin oxidoreductase ctnB (83% amino acid identity); suggesting that the genes involved in OTA production in P. verrucosum may be very similar to those involved in citrinin production in M. anka.