The interaction between tetracycline and the archaeal elongation factor 1α from Sulfolobus solfataricus was investigated. The effects produced by this eubacterial antibiotic indicated that this interaction involved the G-domain of the elongation factor 1α from S. solfataricus, although also the M-domain was required. In fact, in the presence of the antibiotic, an increase in the fluorescence quantum yield of the aromatic region was observed for elongation factor 1α from S. solfataricus and its truncated form lacking the C-terminal domain, but not for that lacking also the M-domain. The increase in quantum yield was restored when the G-domain of elongation factor 1α from S. solfataricus was fused to the M and the C-domains of the eubacterial analogue elongation factor Tu. Tetracycline inhibits protein synthesis catalysed by elongation factor 1α from S. solfataricus; this is accompanied by an increase in the GDP/GTP exchange rate and a slight inhibition of the intrinsic GTPase, suggesting that a main effect of the antibiotic was exerted on the GTP-bound form of the enzyme. Furthermore, the mixed inhibition observed for GTPase confirmed that the interaction, besides the G-domain, involved also other region(s) of elongation factor 1α from S. solfataricus. These results can be useful for studying potential side effects arising from the interaction between tetracycline and eukaryotic elongation factors.