Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Delta(8)-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy-trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Delta(8)-sphingolipid desaturases of S. kluyveri and K. lactis.