The gene coding for ADP-ribosylatable elongation factor 2 (EF-2) from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been cloned and its sequence is reported. Amino acid sequence comparisons showed that EF-2 from S. acidocaldarius is more closely related to eukaryotic EF-2 than to eubacterial EF-G. Consensus sequences are derived from comparison of a region around the unique amino acid diphthamide, which is the target for ADP-ribosylation by diphtheria toxin in archaebacteria and eukaryotes. The conserved positions are likely to constitute a recognition site for the toxin and the histidine-modifying enzymes. A single transcript of approximately the size of the EF-2 gene was observed in Northern blot experiments. Transcription initiation and termination signals were identified in the immediate vicinity of the respective translation start and stop codons of the gene. These results indicate that, in contrast to all prokaryotic EF-2 genes studied previously, the gene of S. acidocaldarius is not located within the streptomycin operon but is transcribed separately.