Analysis of the promoter region of the highly expressed Aspergillus nidulans gpdA gene is described. The nucleotide (nt) sequence of a 1.3-kb region upstream from the ATG was determined. Comparison with promoter regions of other Aspergillus and Neurospora genes revealed several regions of similar sequence. Both random and site-specific mutations were introduced into the promoter region of the gpdA gene, and the resulting mutant promoters were fused to the Escherichia coli lacZ gene. The constructed fusions were introduced into A. nidulans and transformants that contained one copy of these fusions at the argB locus were analysed. beta-Galactosidase assays and primer extension experiments were used to identify sequence elements involved in transcription activation and transcription initiation. Two elements, located around 650 and 250 nt upstream from the major transcription start point (tsp), were identified as transcription activation elements. These elements coincide with regions of putative secondary structure (direct or inverted repeats). A third element, a C + T-rich region directly upstream from the major tsp, was shown to be involved in correct initiation of transcription.