A single protein of Mr 17,000-19,000 and pI approximately equal to 5.1, found in all animal cells we have studied to date, undergoes post-translational modification in growing cells to form the unusual amino acid hypusine. Because of the association of this modification with the increasing rate of protein synthesis during lymphocyte growth stimulation, its subcellular distribution, and its widespread occurrence and structural conservation among animal cells, we considered the possibility that this protein might be a translation initiation factor. Purified rabbit reticulocyte factors (eukaryote initiation factors) eIF-4C and eIF-4D were chosen for study because of their Mr (17,000-19,000) and acidic pI. The hypusine-containing protein and purified eIF-4D showed identity of electrophoretic mobility in both isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis dimensions, while eIF-4C was clearly nonidentical. Purified eIF-4D contained approximately 1 mol of hypusine per mol of protein. Since only one protein has thus far been observed to contain hypusine, we conclude that eIF-4D is the hypusine-containing protein. On the basis of relative synthesis among lymphocyte proteins and detection by Coomassie blue staining, we also conclude that eIF-4D is a major cell protein. It is possible that the activity of this factor is modulated by It is possible that the activity of this factor is modulated by post-translational hypusine formation, which may play a role in regulation of protein synthesis during lymphocyte growth stimulation.