A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen. The NS5A interactor was identified as the alpha subunit of bovine translation elongation factor 1A (eEF1A). Cell-free binding studies were performed with chimeric NS5A fused to glutathione S-transferase (GST-NS5A) expressed in bacteria. GST-NS5A bound specifically to both in vitro-translated and mammalian cell-expressed eEF1A. Moreover, purified eEF1A bound specifically to GST-NS5A attached to a solid phase. Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes. NS5A from all BVDV strains tested so far interacted with eEF1A. The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.