Mutants doubly deficient in phosphoenolpyruvate carboxykinase (pck) and phosphoenolpyruvate synthetase (pps) were unable to grow with succinate as the sole carbon source. A number of pck mutations isolated from pps strains by penicillin selection mapped at 74 min on the Escherichia coli chromosome, between glpD and aroB. Several of the strains containing these mutations had a protein antigenically related to phosphoenolpyruvate carboxykinase, and therefore, the mutations probably represented mutations in the structural gene for this enzyme. Phosphoenolpyruvate carboxykinase was regulated at the level of transcription by catabolite repression. Enzyme levels also increased in stationary-phase cultures by a mechanism independent of cyclic adenosine monophosphate or the product of the relA gene.