A procedure was established for the rapid isolation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (PEPCK) from an overproducing strain. Overexpression was achieved by the transformation of yeast cells with the multicopy plasmid YEp352 harbouring the PEPCK structural gene. The enzyme was purified to homogeneity using first anion-exchange chromatography on Q-Sepharose followed by hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration on Sephacryl S200. The purified phosphoenolpyruvate carboxykinase was further characterized with respect to the molecular mass, displaying an apparent molecular mass corresponding to a tetrameric form.