When normal human peripheral lymphocytes are treated with mitogen and grown in the presence of [3H]putrescine or [terminal methylenes-3H]spermidine, label is incorporated predominantly into one cellular protein. The radioactive constituent of this protein was identified as the unusual amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This was accomplished by isolation of the component from proteolytic digests or acid hydrolysates and comparison with authentic hypusine by chromatography, conversion to the 2,4-dinitrophenyl derivative, and oxidative degradation. The observed relationships among intracellular levels of labeled putrescine, polyamines, and protein bound hypusine after growth of cells with the various labeled amines and with or without an inhibitor of polyamine biosynthesis supply evidence that spermidine is the immediate amine precursor of hypusine and that the 4-amino-2-hydroxybutyl portion of hypusine derives from the butylamine moiety of spermidine.