Filamentous fungi are considered an attractive resource for the discovery and production of bioactive compounds. To facilitate molecular breeding, biosynthetic genes must be rapidly identified. But, even after the chemical structure of a compound is identified, finding the corresponding biosynthetic genes in the fungal genome still remains a challenge. In an attempt to overcome this difficulty and to easily characterize each gene in a cluster, we constructed a heterologous expression system using Aspergillus oryzae. The approximate region covering the citrinin biosynthetic gene cluster from Monascus purpureus was introduced into A. oryzae via an Aspergillus-Escherichia coli shuttle cosmid vector without manipulating the gene structure. The transformants showed slight but reproducible citrinin production and definite transcription of the biosynthetic genes. Introducing additional copies of an activator gene (ctnA), controlled by the Aspergillus nidulans trpC promoter, into the citrinin-cluster-containing transformants enhanced the transcription of all the genes in the cluster and resulted in an almost 400-fold higher citrinin production compared to that of the parental transformant. This result suggested that CtnA controlled citrinin production in this system in the same way as in the native strain. This is the first report documenting the heterologous production of functional fungal secondary metabolites in A. oryzae following the introduction of an entire gene cluster.