SummaryIn Aspergillus nidulans, proline can serve both as a carbon and a nitrogen source. The transcription of the prnB gene, encoding the proline transporter, is efficiently repressed only by the simultaneous presence of ammonium and glucose. Thus, repression of this gene demands the activation of the CreA repressor and the inactivation of the positive‐acting GATA factor AreA. Repression of all other prn structural genes results largely from inducer exclusion. In an areA null mutation background, prnB is repressible by the sole presence of glucose. We have determined by EMSA and missing‐base interference experiments that there are 15 AreA‐binding sites in the prnD‐prnB intergenic region. Only sites 13/14, in the proximity of the prnB TATA box, are clearly involved in transcriptional activation and regulation. Mutation of these sites mimics qualitatively the regulatory effect of an areA null mutation. The deletion of the TATA box has a measurable effect on the maximal level of prnB transcription but does not alter the regulation pattern of this gene.