Responses of Escherichia coli to chemical stimuli are mediated by a family of sensory transducer proteins. These transmembrane proteins detect stimuli and convey sensory information to the flagella. Behavioral adaptation to environmental changes is correlated with the reversible methylation of the transducer proteins on several (4 to 6) specific glutamic acid residues to form methyl esters. We have assayed the activity of the chemotaxis-specific methylesterase, the product of the cheB gene, by measuring the product of the demethylation reaction, methanol, using a modification of a previous method (Toews, M.L., Goy, M.F., Springer, M.S., and Adler, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5544-5548; Terwilliger, T.C., Bogonez, E., Wang, E.A., and Koshland, D. E., Jr. (1983) J. Biol. Chem. 258, 9608-9611). In this study, we establish the use of a sensitive flow apparatus for measuring stimulus-induced changes in methylesterase activity of intact E. coli cells. Responses to positive and negative stimuli consist of transient decrease and increases in methylesterase activity, respectively. In addition, chase experiments demonstrated that all assayable methyl groups are nearly equivalent. The results are not consistent with the view that the methyl groups put on the transducer proteins as a result of a positive stimulus are the first to be removed when that stimulus is withdrawn. It seems more likely that recently added methyl groups become part of a pool of kinetically equivalent members, any of which can be removed when the stimulus is withdrawn. The flow measurements provide a powerful and simple biochemical assay that complements behavioral studies of chemotaxis.