5-(N-Ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, reduced [35S]methionine incorporation in proteins and induced the phosphorylation of a Mr 95,000 protein in bovine aortic endothelial cells. This protein was previously shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acetate nor inhibited by ammonium chloride, and it was reproduced by 2',4'-dimethylbenzamil, an analog of amiloride that is inactive on the Na+/H+ antiport. Furthermore, EIPA enhanced the Ca2(+)-dependent phosphorylation of a similar Mr 95,000 protein in a cell-free system, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has already been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride analogs inhibit protein synthesis.