Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the first step in hypusine biosynthesis. Amino acid sequences of five tryptic peptides from rat deoxyhypusine synthase were found to match partially the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061). In order to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase,a 1.17-kilobase pair cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S. cerevisiae cDNA by polymerase chain reaction and was expressed in Escherichia coli B strain BL21 (DE3). The recombinant protein was found mostly in the E. coli cytosol fraction and comprised approximately 20% of the total soluble protein. The purified form of the expressed protein effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A precursors as well as in human precursor and in those from Chinese hamster ovary cells. The molecular mass of the enzyme was estimated to be 172,000 +/- 4,300 Da by equilibrium centrifugation. The mass of its polypeptide subunit was determined to be approximately 43,000 Da, in close agreement with that calculated for the coding region of the YHRO68w gene. These findings show that this gene is a coding sequence for yeast deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.