The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.