The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5 and 3 flanking sequences, was determined. The structural part of the A. niger pkiA gene is 2054 bp long and is interupted by seven putative introns. Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58 130 Da. Extensive homology is found with pyruvate kinase from A. nidulans; only 33 amino acids are different between both proteins. Transformation experiments using the pyr A gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase. Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus. Predominantly co-integration of the pyrA-and the pkiA-containing plasmids was found in the cases examined.