An open reading frame (ORF) was found upstream of the mdh gene in Thermus flavus by computer-aided analysis. It was identified as the gene encoding the alpha subunit of succinyl-CoA synthetase (SCS) and termed scsA. Nucleotide sequencing of a further upstream region revealed the presence of another ORF, corresponding to the sequence of the beta subunit of SCS. The latter gene was termed scsB. The scsB gene was found to contain an unusual translational initiation codon, TTG. S1 nuclease mapping indicates that transcription starts at the nucleotide at position--31 upstream of the initiation codon of the beta gene. The scsB and scsA genes along with the mdh gene appear to form an operon and are most likely co-transcribed in this order, because the intercistronic regions between them are very short; in fact, the termination codon of scsB overlaps the initiation codon of scsA. A stretch characteristic of the--10 region of a typical prokaryotic promoter was found upstream of scsB, whereas no sequence characteristic of a typical--35 region was present. Escherichia coli harboring a plasmid containing scsA and scsB did not produce thermostable SCS activity, even when a synthetic promoter for E. coli was attached. However, when an inverted repeat present in front of scsB, which covers the putative ribosome-binding site and is capable of forming a stable stem-loop structure, was altered by site-directed mutagenesis, overproduction of heat-stable SCS was observed.