The cloned X-bacterial gene (groEx) which is analogous to groE of E. coli strongly expressed in E. coli when grown at the temperature 27°C or higher without having to add any inducers. By S1-nuclease mapping, primer extension analysis and site directed mutagenesis, we found 4 promoters in this gene. Among them two promoters located at 5′-extended region of the gene are homologous to the promoters found in groE family of heat-shock genes; they are, σ32 factor-dependent P1 promoter and σ70 factor-dependent P2 promoter. The other two promoters found within the coding region of groESx were P3, 5′-TTGGCG-(18 bases)-AATACT-3′ and P4, 5′-TTGGCA-(19 bases)-TAAAGT which overlapped within 49 bases. These unique intragenic σ70-dependent promoters are the first to be cloned and characterized in groE analogous heat-shock genes so far. These P3 and P4 promoters appeared to be responsible for the strong expression of GroELx in X-bacteria in vivo.