Fungi, like plants, are capable of producing the 18-carbon polyunsaturated fatty acids linoleic acid and alpha-linolenic acid. These fatty acids are synthesized by catalytic reactions of Delta12 and omega3 fatty acid desaturases. This paper describes the first cloning and functional characterization of a yeast omega3 fatty acid desaturase gene. The deduced protein encoded by the Saccharomyces kluyveri FAD3 gene (Sk-FAD3) consists of 419 amino acids, and shows 30-60 % identity with Delta12 fatty acid desaturases of several eukaryotic organisms and 29-31 % identity with omega3 fatty acid desaturases of animals and plants. During Sk-FAD3 expression in Saccharomyces cerevisiae, alpha-linolenic acid accumulated only when linoleic acid was added to the culture medium. The disruption of Sk-FAD3 led to the disappearance of alpha-linolenic acid in S. kluyveri. These findings suggest that Sk-FAD3 is the only omega3 fatty acid desaturase gene in this yeast. Furthermore, transcriptional expression of Sk-FAD3 appears to be regulated by low-temperature stress in a manner different from the other fatty acid desaturase genes in S. kluyveri.