The amino acid hypusine is formed by post-translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF-5A has been known to contain this unique component. We isolated and purified a hypusine-containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius. The mainly cytosolic protein comprised about 0.03% of the post-ribosomal supernatant protein. No other hypusine-containing protein could be detected in S. acidocaldarius. The molar ratio of hypusine/hypusine-containing protein was 1:1. SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF. The N-terminus was blocked. Four cyanogen bromide fragments were partially sequenced and used to derive two 17-base oligonucleotide probes. A 3-kb HindIII fragment of genomic DNA hybridizing with both probes was cloned. By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa. It contained all cyanogen bromide sequences analysed. Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine-containing protein were identical to the nonapeptides centered by hypusine in the three eIF-5A proteins sequenced so far. The Edman procedure gave no phenylthiohydantoin derivative for this position. For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted. In addition, there were a number of conservative changes. The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine-containing protein and eIF-5A. Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function.