The biosynthesis of penicillin, a secondary metabolite produced by Penicillium chrysogenum, is subject to sophisticated genetic and metabolic regulation. The structural genes, pcbC and pcbAB, which encode two of the penicillin biosynthetic enzymes are separated by a 1.16-kb intergenic region and transcribed divergently from one another. To identify and characterize nuclear proteins which interact with the pcbAB-pcbC intergenic promoter region, crude and partially purified nuclear extracts were used in mobility shift and DNA footprinting assays. Multiple DNA-binding proteins appear to bind to different regions of this DNA segment. An abundant nuclear protein, nuclear factor A (NF-A), binds at a single site in the intergenic promoter region and recognizes an 8-bp sequence, GCCAAGCC. Penicillin production is sensitive to nitrogen catabolite repression. The global-acting nitrogen regulatory protein NIT2 of Neurospora crassa binds strongly to the intergenic promoter region of the pcbAB and pcbC genes at a single site that contains two closely spaced GATA sequences.