A plasmid vector (pDEL2) was engineered for the purpose of introducing a deletion within the aflatoxin (AF) biosynthetic gene cluster of Aspergillus parasiticus. The vector was constructed by PCR amplification of a region of the AF gene cluster from an A. parasiticus isolate that had undergone an aberrant recombinational event during transformation with a nor A-niaD gene disruption vector. This recombinational event resulted in the deletion of an approximately 6-kb region of the AF gene cluster and accumulation of the AF precursor averantin (AVN). Northern hybridization analysis confirmed that the deletion event resulted in no detectable transcription of the norA gene or the AF biosynthetic genes, avnA, verA, and ver-1. Transformation of A. parasiticus RHN1 with pDEL2 resulted in 16% of the transformants accumulating AVN. Southern hybridization analysis of randomly selected AVN-accumulating transformants indicated that all had undergone a double-crossover homologous, recombinational event resulting in the 6-kb norA to avnA deletion within the AF gene cluster. Aflatoxin precursor feeding studies performed on one of the AVN-accumulating, RHN1(pDEL2) transformants confirmed that the enzyme activities associated with the deleted genes were no longer present.