GTP-binding protein/transglutaminases (tissue transglutaminases or TGases) have been implicated in a variety of cellular processes including retinoic acid (RA)-induced apoptosis. Recently, we have shown that RA activates TGases as reflected by stimulated GTP binding, increased membrane association, and stimulated phosphoinositide lipid turnover. This prompted us to search for cellular proteins that bind TGases in a RA-stimulated manner. In this report, we show that the eukaryotic initiation factor (eIF-5A), a protein that is essential for cell viability, perhaps through effects on protein synthesis and/or RNA export, associates with the TGase in vivo. The interaction between eIF-5A and TGase is specific for the GDP-bound form of the TGase and is not detected when the TGase is pre-loaded with GTP gamma S. The TGase-eIF-5A interaction also is promoted by Ca2+, Mg2+, and RA treatment of HeLa cells. In the presence of retinoic acid, millimolar levels of Ca2+ are no longer required for the TGase-eIF-5A interaction. Nocodazole treatment, which blocks the cell cycle at mitosis (M phase), strongly inhibits the interaction between eIF-5A and cytosolic TGase. The interaction between TGase and eIF-5A and its sensitivity to the nucleotide-occupied state of the TGase provides a potentially interesting connection between RA signaling and protein synthesis and/or RNA trafficking activities.