Analysis of the promoter of the penicillin biosynthesis aat (penDE) gene of Aspergillus nidulans using band-shift assays led to the identification of a CCAAT-containing DNA element which was specifically bound by a protein (complex). The identified DNA element was localised about 250 bp upstream of the transcriptional-start sites of aat. Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat-lacZ gene fusion. The identified binding site thus was functional in vivo and positively influenced at expression. Partial purification of the CCAAT binding protein and cross-competition experiments provided evidence that the binding protein is identical to the identified putative penicillin-regulatory protein PENR1, binding to the CCAAT element in the bidirectional intergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENR1 seems to be involved in the regulation of all three penicillin-biosynthesis genes. Cross-competition experiments demonstrated that the promoter region of the corresponding aat (penDE) gene of Penicillium chrysogenum was capable to dilute the shift of the A. nidulans probe with PENR1, suggesting the presence of a similar regulatory mechanism in this fungus. Taken together with previous data, CCAAT-containing DNA elements thus seem to represent major cis-acting sites in the promoters of beta-lactam-biosynthesis genes.