The control of isocitrate dehydrogenase through phosphorylation is necessary for growth of Escherichia coli on acetate as the sole carbon source. To understand the mechanism by which phosphorylation inactivates isocitrate dehydrogenase, the sequence of icd, the isocitrate dehydrogenase structural gene, was determined and this information was used to construct mutants at the site of phosphorylation. Introduction of a negatively charged aspartate for the serine that is phosphorylated completely inactivates isocitrate dehydrogenase. Substitution of the serine with other amino acids results in a partially active enzyme in which both maximal velocity and interaction with substrates has been altered. Neither threonine nor tyrosine, when substituted for the serine at the phosphorylation site, is detectably phosphorylated by isocitrate dehydrogenase kinase.