Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY-P, is important for preparing bacteria and archaea to respond sensitively to new stimuli. In an extension of previous work (Szurmant, H., Bunn, M. W., Cannistraro, V. J., and Ordal, G. W. (2003) J. Biol. Chem. 278, 48611-48616), we describe a new family of CheY-P phosphatases, the CYX family, that is widespread among the bacteria and archaea. These proteins provide another pathway, in addition to the ones involving CheZ of the gamma- and beta-proteobacteria (e.g. Escherichia coli) or the alternative CheY that serves as a "phosphate sink" among the alpha-proteobacteria (e.g. Sinorhizobium meliloti), for dephosphorylating CheY-P. In particular, we identify CheC, known previously to be involved in adaptation to stimuli in Bacillus subtilis, as a CheY-P phosphatase. Using an in vitro assay used previously to demonstrate that the switch protein FliY is a CheY-P phosphatase, we have shown that increasing amounts of CheC accelerate the hydrolysis of CheY-P. In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY-P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY-P. CheC appears to be primarily involved in restoring normal CheY-P levels following the addition of attractant, whereas FliY seems to act on CheY-P constitutively. The activity of CheC is relatively low compared to that of FliY, but we have shown that the chemotaxis protein CheD enhances the activity of CheC 5-fold. We suggest a model for how FliY, CheC, and CheD work together to regulate CheY-P levels in the bacterium.