Genomic and cDNA clones encoding glucose-6-phosphate dehydrogenase (G6PD) were isolated from the fungusAspergillus niger. Sequence analysis of the glucose-6-phosphate dehydrogenase gene (gsdA) revealed an open reading frame of 1530 bp, encoding a protein of 58,951 kDa. The (gsdA) gene is interrupted by nine introns the most proximal of which is exceptionally large (348 bp). The region upstream of the ATG contains several C + T-rich stretches. The two major and one minor transcription start points are all located within these regions. In the upstream region several direct and inverted repeats, but no clear TATA or CCAAT boxes can be found.A. niger strains overproducing G6PD were constructed by cotransformation of (gsdA) subclones. Overexpression of G6PD was shown to be deleterious for the fungus, especially when cotransformants were grown on media containing ammonia. Attempts to construct a (gsdA) null mutant by gene disruption were unsuccessful.