A putative ochratoxin A (OTA) biosynthetic gene cluster inP. nordicum has been identified. The first part of the gene cluster is located on a DNA fragment of 10 kb in length and harbours three genes. A gene with high homology to an alkaline serine protease gene (accession number AY557343), which represents the upstream border of the cluster. Furthermore the fragment carries a large part (about 2 kb) of the 5' end of a polyketide synthase (otapksPN, accession number AY196315) and a complete non-ribosomal peptide synthetase (otanpsPN, accession number AY534879). The second part of the cluster is located on a 4.3 kb fragment that harbours three open reading frames (ORFs) encoding putative OTA biosynthetic proteins: one incomplete ORF at the 5' end of the fragment demonstrated homology to an organic anion transporter from rat kidneys (otatraPN). This transporter has been described to be responsible for the transport of toxic OTA out of the cell. One complete ORF of 951 nucleotides is also located on this fragment. This gene has limited homology to a chloroperoxidase fromGluconobacter oxidans. At the 3' end of this DNA fragment is an incomplete open reading frame of a potential nitrate transporter. The transcription of all putative OTA biosynthetic genes is increased under OTA conducive conditions. The expression kinetics of the genes resembles that of secondary metabolite biosynthetic genes, in which these genes are co-ordinately expressed during the late growth phase.PCR analysis demonstrated that the gene cluster is only present in the two ochratoxin A producingPenicillium species,P. verrucosum andP. nordicum. P. nalgiovense, a species occurring in the same habitat asP. nordicum carries inactive homologues of the genes. All other species proved to be negative for the genes. This was also true for OTA producing Aspergilli.