Gene libraries of DNA from an n-alkane-assimilating yeast strain, Candida maltosa IAM12247, were constructed, using Escherichia coli plasmid vector pBR322. A LEU gene from C. maltosa was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation, while in the other orientation, it was expressed only weakly. In S. cerevisiae, the Candida LEU gene was efficiently expressed when inserted into a shuttle vector pRC3 in both orientations, suggesting that the isolated Candida DNA fragment contains a promoter sequence of Candida in front of the LEU gene, which is operative in S. cerevisiae but not in E. coli. In addition, our data suggest that the cloned LEU fragment also contains an ARS (autonomously replicating sequence) site of C. maltosa.