Eukaryotic initiation factor 5A (eIF‐5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF‐5A gene is essential for cell survival and proliferation, the precise function and localization of eIF‐5A remain unclear. In this study, we have determined the subcellular distribution of eIF‐5A by indirect immunofluorescent staining and by direct visualization of green fluorescent protein tagged eIF‐5A (GFP‐eIF5A). Immunofluorescent staining of the formaldehyde‐fixed cells showed that eIF‐5A was present in both the nucleus and cytoplasm. Only the nuclear eIF‐5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF‐5A in living cells revealed the same whole‐cell distribution pattern. However, a fusion of an additional pyruvate kinase (PK) moiety into GFP‐eIF‐5A precluded the nuclear localization of GFP‐PK‐eIF‐5A fusion protein. Fusion of the GFP‐PK tag with three different domains of eIF‐5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor‐mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP‐Rev, but not of GFP‐eIF‐5A. The whole‐cell distribution pattern of eIF‐5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1‐dependent nuclear export. Collectively, our data indicate that eIF‐5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling. J. Cell. Biochem. 86: 590–600, 2002. © 2002 Wiley‐Liss, Inc.