The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous 18kDa protein of eukaryotic and archaeal organisms, which undergoes a unique post-translational modification wherein a specific lysine residue is converted into hypusine. In eukaryotes eIF5A is described to be localized to the cytoplasm, either associated with ribosomes or in the perinuclear region, as well as to the nucleus. Based on multiple sequence alignment analysis of eIF5A proteins from different eukaryotic organisms and some of their orthologous counterparts from archaea, we recognized that the former sequences have an extended N-terminal segment. Considering that a pivotal difference between organisms from eukarya and archaea is the presence of intracellular membranes, we carried out experiments to verify any possible roles that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our experimental approach relied upon immunocytochemical and fluorescence localization of various forms of eIF5A in COS-7 cells transiently transfected with expression plasmids containing the corresponding eIF5A constructs. Our results indicate that the N-terminal extension of the eukaryotic eIF5A plays a role in determining preferential nuclear/perinuclear localization of this protein.