A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that, a 1.7 kb BglII-SphI DNA fragment isolated form this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment, was 1.4 kilonucleotides in size. Genetic complementation, proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.