The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.