The beta-lactam antibiotic penicillin is produced as an end product by some filamentous fungi only. It is synthesized from the amino acid precursors L-alpha-aminoadipic acid, L-cysteine, and L-valine. Previous data suggested that certain amino acids play a role in the regulation of its biosynthesis. Therefore, in this study the effects of externally added amino acids on both Aspergillus (Emericella) nidulans penicillin production and expression of the bidirectionally oriented biosynthesis genes acvA (pcbAB) and ipnA (pcbC) were comprehensively investigated. Different effects caused by amino acids on the expression of penicillin biosynthesis genes and penicillin production were observed. Amino acids with a major negative effect on the expression of acvA-uidA and ipnA-lacZ gene fusions, i.e., histidine, valine, lysine, and methionine, led to a decreased ambient pH during cultivation of the fungus. An analysis of deletion clones lacking binding sites for the pH-dependent transcriptional factor PACC in the intergenic regions between acvA-uidA and ipnA-lacZ gene fusions and in a pacC5 mutant (PacC5-5) suggested that the negative effects of histidine and valine on acvA-uidA expression were due to reduced activation by PACC under acidic conditions. These data also implied that PACC regulates the expression of acvA, predominantly through PACC binding site ipnA3. The repressing effect caused by lysine and methionine on acvA expression, however, was even enhanced in one of the deletion clones and the pacC5 mutant strain, suggesting that regulators other than PACC are also involved.