Translation initiation factor eIF-5A (previously named eIF-4D) is an essential and highly conserved protein in eukaryotic cells that promotes formation of the first peptide bond. One of its lysine residues is post-translationally modified by spermidine to form hypusine, a unique residue required for eIF-5A activity. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B. The two genes are regulated reciprocally by oxygen, where under aerobic conditions TIF51A is expressed and TIF51B is repressed, and under anaerobic conditions the opposite occurs. In order to study the products of the two genes individually, yeast strains were constructed that express either TIF51A or TIF51B under control of a galactose promoter. Each gene gives rise to two isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic), both of which carry the hypusine modification. Expression of either TIF51A or TIF51B promotes growth under both aerobic and anaerobic conditions, indicating that the two gene products function indistinguishably. The human cDNA encoding eIF-5A also was expressed in yeast, and the plasmid shuffle technique was used to demonstrate that the human protein can substitute for the homologous yeast protein in vivo. These results indicate that human and yeast eIF-5A are not only conserved at the sequence level but are functionally interchangeable in vivo.