Rv2027c is a putative orphan histidine sensor kinase that bears strong homology to DevS of the hypoxia-responsive DevR-DevS two-component system in M. tuberculosis. The cytosolic C-terminal domain of Rv2027c protein (Rv2027c 194 ) was overexpressed in E. coli and biochemically characterized. Rv2027c 194 underwent autophosphorylation at a conserved His 392 residue and engaged in phosphotransfer with DevR response regulator. The rates of autophosphorylation and the stabilities of the phosphorylated species were broadly similar in Rv2027c and DevS. However, unlike DevS, Rv2027c utilized Ca 2þ as an alternative divalent ion during autophosphorylation. In contrast to DevS which completed phosphotransfer to DevR in 5-10 min, phosphotransfer from Rv2027c$P was only partial at 30 min. Unlike devS transcription that was hypoxia-responsive, Rv2027c transcript levels were not upregulated from basal levels during hypoxia. The differential regulation of devS and Rv2027c genes, the ability of Rv2027c to utilize Ca 2þ as a divalent cation in autophosphorylation at physiological concentrations and to engage in phosphotransfer with DevR suggests that the DevR regulon could be modulated by more than one environmental cue relayed through DevS and Rv2027c.