Regulation of the expression of the afp gene that codes for the Antifungal Protein of Aspergillus giganteus was investigated using a reporter system. For this purpose, the E. coli reporter gene uidA encoding beta-glucuronidase (GUS) was placed under the control of the afp promoter. No homologous integration of the reporter construct into the afp site was observed among 156 transformants tested. In one of the transformants carrying a single, ectopically integrated, copy of the construct, GUS and AFP both displayed exactly the same temporal expression patterns under various cultivation conditions, as assayed by Northern and protein analyses. Thus, this transformant was used to identify factors that are involved in the transcriptional regulation of afp expression. Expression is only detectable in the vegetative mycelium, whereas no expression occurs in aerial hyphae or conidia, indicating that afp expression is developmentally regulated. Transcription of afp is regulated by ambient pH, being suppressed under acidic conditions and strongly induced under alkaline conditions. This observation suggests that PacC regulates the afp gene, which is consistent with the presence of two putative PacC binding sites within the 5' upstream region. Transcription is not subject to carbon catabolite repression or nitrogen metabolite repression. The expression of afp is up-regulated by heat shock, upon growth in the presence of excess NaCl and ethanol, and under conditions of carbon starvation. In contrast, expression decreases slightly in the presence of hydrogen peroxide and under nitrogen starvation. These data are compatible with the presence of a putative heat shock element (NTTCNNGANTTCN) and five putative C(4)T stress-responsive elements within the afp promoter.