Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly regulated. The response regulator RegX3 and the cytoplasmic portion of the histidine kinase SenX3 from Mycobacterium bovis BCG were overproduced in Escherichia coli and purified as N-terminally (His)(6)-tagged proteins. Phosphorylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved histidine and aspartate residues, respectively. In addition, as shown by electrophoretic mobility shift assays, (His)(6)RegX3 was able to specifically bind to the promoter region of the senX3-regX3 operon. Furthermore, operon fusion analyses indicated that the overexpression of the senX3-regX3 operon increases the activity of the senX3 promoter in Mycobacterium smegmatis. Together, these results indicate that the mycobacterial SenX3-RegX3 two-component system is positively autoregulated.