The carbon storage regulator gene csrA has been shown previously to dramatically affect the biosynthesis of intracellular glycogen in Escherichia coli through its negative control of the expression of two glycogen biosynthetic operons and the gluconeogenic gene pckA (Romeo, T., Gong, M., Liu, M. Y., and Brun-Zinkernagel, A. M. (1993) J. Bacteriol. 175, 4744-4755). Examination of the effects of csrA on several enzymes, genes, and metabolites of central carbohydrate metabolism now establishes a more extensive role for csrA in directing intracellular carbon flux. Phosphoglucomutase and the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate synthetase were found to be under the negative control of csrA, and these enzyme activities were maximal during the early stationary phase of growth. The enzymes glucose-6-phosphate isomerase, triose-phosphate isomerase, and enolase were positively regulated by csrA. Thus, csrA exerts reciprocal effects on glycolysis versus gluconeogenesis and glycogen biosynthesis. The glycolytic isozymes pyruvate kinase F and A (encoded by pykF and pykA, respectively) and phosphofructokinase I and II (pfkA and pfkB, respectively) exhibited differential regulation via csrA. Since the individual members of these isozyme pairs are allosterically regulated by different cellular metabolites, csrA is also capable of fine-tuning the allosteric regulation of glycolysis. In contrast, the expression of genes of the pentose phosphate pathway was weakly or negligibly affected by csrA.