The soil bacterium Rhizobium meliloti responds to chemotactic stimuli by modulating the rotary speed of its flagella. Unlike in Escherichia coli, the signal transduction chain of R. meliloti contains two different response regulators, CheY1 and CheY2, but no CheZ phosphatase. Phosphorylation of CheY1 and CheY2 by the central ATP-dependent autokinase, CheA, is the crucial step in signal transduction. In vivo, phospho-CheY2 (CheY2-P) is the chief regulator of flagellar rotation, its action being modulated by CheY1 [Sourjik, V., and Schmitt, R. (1996) Mol. Microbiol. 22, 427-436]. In this study, we have investigated these phosphotransfer reactions in vitro using the radiolabeled recombinant proteins, CheA (labeled via [gamma-32P]ATP), CheY1, and CheY2 (labeled via acetyl [32P]phosphate). Our results are consistent with the following four-step phosphotransfer: (i) ATP-dependent autophosphorylation of CheA (with a limiting rate constant of 0.008 s-1 at saturating ATP concentrations); (ii) rapid phospho transfer from phospho-CheA to CheY1 and CheY2; (iii) autodephosphorylation of CheY1-P and CheY2-P with half-lives of 12 +/- 1 s and 10.5 +/- 1 s, respectively; and (iv) reversible phosphotransfer from CheY2-P to CheA. In the three-component mixture, CheA/CheY1/CheY2, we observed rapid phosphotransfer from CheY2-P via CheA to CheY1. Thus, CheY1 assumes the role of a "phosphatase" of CheY2-P by acting as a sink for phosphate, whenever unphosphorylated CheA is present. The intracellular concentrations of CheA/CheY1/CheY2 determined immunochemically were 1.5 microM:20 microM:20 microM, a range that was adopted for in vitro assays. The results reflect a unique control by CheY1 of the active, phosphorylated state of the main response regulator, CheY2-P. This mechanism appears to be a new twist to signal transduction among members of the alpha-subgroup of proteobacteria.