Eukaryotic translation initiation factor 5A (eIF-5A, older nomenclature, eIF-4D) is a highly conserved protein that contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The biosynthesis of hypusine occurs posttranslationally in only this protein by modification of a single lysine residue (Lys50 in the human eIF-5A precursor). The basis for the specificity of this modification with respect to the substrate protein was investigated using fragments of eIF-5A precursor protein, each containing this lysine residue, as substrates for deoxyhypusine synthase, the first enzyme in hypusine synthesis. Proteolytic fragments (5-6 kDa) of ec-eIF-5A (the precursor form of eIF-5A produced in Escherichia coli by expression of the human eIF-5A cDNA) generated by specific cleavage by endoproteinases Arg-C, Asp-N, or Glu-C, did not act as substrates for deoxyhypusine synthesis. A series of truncated forms of the eIF-5A precursor protein generated by expression in E. coli of recombinant deletion constructs from the human eIF-5A cDNA were tested. Truncation of up to 9 amino acid residues (Met1-Thr9) from the NH2 terminus or 64 amino acid residues (Leu91-Lys154) from the COOH terminus did not significantly decrease the substrate reactivity, but removal of an additional 10 amino acids from either side did. Deletion of 34 amino acid residues (Met1-Lys34) from the NH2 terminus or of 84 amino acid residues (Asp71-Lys154) from the carboxyl terminus caused complete loss of substrate property. The results obtained thus far define the minimum domain of the eIF-5A precursor protein required for enzymatic deoxyhypusine synthesis as Phe30-Asp80, which corresponds to a region of high amino acid conservation in this protein throughout the eukaryotic kingdom.