The isolation and characterization of the highly expressed glyceraldehyde-3-phosphate dehydrogenase (GPD)-coding gene (gpdA) of Aspergillus nidulans is described. The gene was isolated from an A. nidulans lambda gene library with a Saccharomyces cerevisiae GPD-coding gene as a probe. Unlike many other eukaryotes, A. nidulans contains only one GPD-coding gene. At the amino acid level, homology with other GPD enzymes is extensive. The A. nidulans gene contains seven introns, one of which is positioned in the 5'-untranslated part of the gene. The major transcription start point is found at 172 bp upstream from the start codon. Polyadenylation occurs at several sites about 200 bp downstream from the stop codon. Comparison of 5' and 3' flanking sequences with flanking sequences of other highly expressed (glycolytic) genes shows several regions of similar sequence.