Hypusine formation on an 18,000-dalton cellular protein is a unique spermidine-dependent, post-translational modification that appears to be ubiquitous in mammalian cells. To determine whether this modification also exists in lower eukaryotes, we examined possible labeling in vitro and in vivo of cellular protein(s) by [3H]spermidine in a mutant strain of Neurospora crassa (arge-12 ota aga) in which ornithine and polyamine synthesis could be nutritionally manipulated. Because of poor uptake of polyamines in this organism, [3H]ornithine, the immediate precursor of polyamines, was used for the in vivo labeling experiment. Both in vitro and in vivo labeling resulted in a specific labeling of a 21,000-dalton protein. Radioactive hypusine was recovered from radiolabeled 21,000-dalton protein following acid hydrolysis. The in vitro labeling of the 21,000-dalton protein was dramatically stimulated by NAD+ and NADP+, but not by FMN or FAD, suggesting that an NAD+/NADP(+)-dependent oxidative cleavage of spermidine is involved in deoxyhypusine formation. Isoelectric focusing/sodium dodecyl sulfate two-dimensional gel analysis revealed three isoforms of the in vitro labeled 21,000-dalton protein, with pI values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo gave only one spot with a pI value of approx. 3.5.