In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.