The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A Mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we, demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis. (C) 2009 Elsevier Inc. All rights reserved.