Deoxyhypusine synthase catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor using spermidine as the substrate. We have purified deoxyhypusine synthase from Neurospora crassa to apparent homogeneity (Tao, Y., and Chen, K. Y. (1995) J. Biol. Chem. 270, 383-386). We have now cloned and characterized the deoxyhypusine synthase cDNA using a reverse genetic approach. Conceptual translation of the nucleotide sequence of the cloned 1258-base pair cDNA revealed an open reading frame containing 353 amino acids with a predicted M(r) of 38,985. The deoxyhypusine synthase cDNA was subcloned into the expression vector pQE60 to produce a 40,000-dalton recombinant protein on SDS-PAGE which exhibited deoxyhypusine synthase activity. A GenBank search showed that the Neurospora deoxyhypusine synthase cDNA possessed significant sequence homology to a previously uncharacterized yeast sequence (accession number U00061 (1994)). The yeast sequence encodes a protein of 387 amino acids that shows 69% of total amino acid identity and 80% of total amino acid similarity to the Neurospora enzyme. Sequence alignment and hydropathy analysis suggest that the yeast sequence represents deoxyhypusine synthase.