Using a dual-label technique in which 3H- and 14C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at C-1 of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the epsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at C-2 in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. Our findings permit suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-aminobutyraldehyde produced by oxidative cleavage of spermidine is coupled with the epsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.