The yeast Yarrowia lipolytica was shown to be sensitive to the aminoglycoside antibiotic hygromycin B. Spontaneous resistants appeared at a frequency of (2-5) x 10(-7) in media containing 100 mg/l drug. In order to develop a new selective marker for the transformation of this yeast, we constructed new plasmids expressing the Escherichia coli hygromycin-resistance gene (hph) under the control of the promoter and terminator sequences of the strongly expressed XPR2 gene of Y. lipolytica. Direct selection of hygromycin-B-resistant transformants on complete medium was very efficient and resulted in transformation frequencies comparable to those observed with conventional auxotrophic markers. This new marker can be used for integrating single copies of plasmid and for gene disruption and provides a convenient tag for genetic studies.