Eukaryotic translation initiation factor 5A (eIF5A) is an essential protein tightly linked to cellular polyaminehomeostasis. It receives the unique spermidine-derivedposttranslational modification hypusine that is necessary for eIF5A's biochemical activity and cellular proliferation. The eIF5A proteinstimulates ribosomal peptidyl-transferase and may be involved in nucleocytoplasmic mRNA transport. Little is known about the molecular genetics of eIF5A. Here we report on the sequence and molecular characterization of human EIF5A2, a novel phylogenetically conserved gene for eIF5A. EIF5A2 stretches over 17 kb and consists of five exons and four introns. It is localized at 3q25–q27, often noted for chromosomal instability in cancers. EIF5A2 is highly expressed in testis and colorectal adenocarcinoma and at moderate levels in the brain, in contrast to the ubiquitously expressed EIF5A1 gene. Two EIF5A2 mRNAs share a 129-nt 5′ UTR and a coding sequence for the 153-amino-acid eIF5AII protein, but possess two alternative 3′ UTRs of 46 and 890 nt that arise through differential polyadenylation. The protein is 84% identical and 94% similar to eIF5AI. Both EIF5A genes are conserved in vertebrates. Our findings lend further support for a specialized gene expression program of polyamine metabolic proteins and regulators that function to maintain polyamine homeostasis at elevated levels during spermatogenesis.