An in vivo disruption–integration vector system for Thermus thermophilus was developed and used for the functional analysis of an evolutionary-related archaeal protein for lysine biosynthesis. In contrast to fungal one, the putative homoaconitase of T. thermophilus consists of two subunits and catalyzes the second and third steps of lysine biosynthesis. ORFs from hyperthermophilic archaeon Pyrococcus horikoshii, PH1726 and PH1724, share a high degree of amino acid identity with the T. thermophilus subunits LysT and LysU, respectively. In the present report, gene encoding the putative small subunit of archaeal homoaconitase, PH1724, was integrated into the lysU locus of T. thermophilus. The archaeal gene was expressed under the control of PslpA promoter and functional analyses were performed. Transformants were able to grow on minimal medium without lysine when PH1724 ORF was integrated, whereas the lysU disruption led to lysine auxotrophy. Chromosomal integration was verified by PCR analysis, and homoaconitase assay showed that the archaeal gene product functions as a small subunit of homoaconitase, possibly by forming a heterodimer with the LysT subunit of T. thermophilus. These results strongly suggest the functional relation of P. horikoshii PH1724 with LysU in the Thermus lysine biosynthetic pathway, together with functional assignment of LysU as small subunit of homoaconitase. In addition, the provided results indicate that archaeal genes products from hyperthermophiles can be studied in a thermophilic eubacterium such as T. thermophilus.