The region containing the RNA polymerase alpha subunit (RNAPalpha) gene (rpoA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Southern hybridization. The gene order in this region is rpl36-rps13-rps11-rps4-rpoA-rpl17, which is identical to that in some other eubacteria. The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 35,013, the amino acid sequence showing 42% identity to that of Escherichia coli (Ec). From the results of comparison of the amino acid sequence and the predicted secondary structure of the C-terminal domain of Tt RNAPalpha (Tt alphaCTD) with those of Ec, the overall folding is expected to be similar. However, amino acid residues Asn268 and Cys269 in Ec alphaCTD, which are essential for its interaction with DNA or regulatory proteins, were replaced by His and Ser, respectively, in Tt alphaCTD. By means of a T7-based expression system in Ec cells, Tt RNAPalpha was overexpressed and purified. The high thermostability of Tt RNAPalpha was demonstrated by the CD spectra.