Aspergillus nidulans synthesizes penicillins as secondary metabolites when grown under certain culture conditions. Broths containing carbon (C) sources that give rise to carbon catabolite repression (CCR) support a much lower antibiotic yield than broths with non-repressing C sources. Steady-state levels of the isopenicillin N synthetase (IPNS) gene transcript are considerably reduced in mycelia grown with repressing C sources and are depressed in mycelia grown with sugars which do not cause CCR, indicating that penicillin biosynthesis is regulated by CCR through transcriptional control of structural genes. CCR is sufficient to explain the temporal window of expression of the IPNS gene during the growth cycle since (i) the transcript becomes derepressed as soon as the concentration of a repressing C source drops to non-repressing levels and (ii) derepressing C sources sustain derepressed IPNS transcription at all tested moments of the growth cycle. Several tested hypofunctional mutations in creA (the negatively acting regulatory gene which mediates CCR in A. nidulans) do not cause full derepression of IPNS transcript in the presence of a repressing C source. The slight degree of IPNS derepression caused by some creAd (derepressed) alleles parallels the strength of the mutation (as determined by the morphological effect they elicit).