We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA- or lysR- genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys- mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys- mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys- and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-gamma-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.