The genes for the patulin biosynthetic pathway are most likely arranged in a cluster, as is often the case for other mycotoxins. With this in mind, GeneWalking has been performed to identify genes both upstream and downstream of the isoepoxydon dehydrogenase (idh) gene. A gene present in Penicillium griseofulvum NRRL 2159A had high sequence homology to the isoamyl alcohol oxidase (iao) gene and was detected downstream of the idh gene and in the same orientation. By virtue of the presence of a signal peptide sequence, the newly identified gene coded for a secreted protein with an FAD-binding domain and potential for N-glycosylation. An open reading frame consisted of 1946 nucleotides, containing four putative introns and encoding a 22 amino acid signal peptide. The 571 amino acid mature protein contained nine cysteine residues and had 11 potential N-linked glycosylation sites. Searches using GenBank indicated that Aspergillus terreus, A. oryzae, A. fumigatus, and Gibberella zeae contain genes coding for a putative isoamyl alcohol oxidase. When the translated query was compared with the translated database, the highest scores were seen with A. clavatus (E value of 0.00), A. fumigatus (E value of 8e(-142)), and A. oryzae and A. terreus (each having an E value of 2e(-141)). Reverse transcription-polymerase chain reaction analysis confirmed that the iao gene was transcribed. The amplified products were sequenced for confirmation of their identities. This is the first report of an isoamyl alcohol oxidase gene in a species of the genus Penicillium.