We present evidence that ribulose-5-phosphate epimerase, a central metabolic enzyme acting in the non-oxidative branch of the pentose-phosphate pathway, is encoded by a gene in the dam containing operon of Escherichia coli. Enzymatic assays confirm that this gene encodes ribulose-5-phosphate epimerase activity. Disruption of the gene (rpe) causes loss of enzymatic activity and renders the rpe mutant unable to utilize single pentose sugars, indicating that rpe supplies the only ribulose-5-phosphate epimerase activity in E. coli. Growth of the rpe mutant is impaired in complex LB medium and severely impaired in minimal medium containing glycolytic carbon sources or gluconate. Enrichment with casamino acids abolishes or strongly relieves growth suppression in minimal medium. Aspartate counteracts the impaired growth in glycolytic carbon sources but not in gluconate. We suggest that the absence of the Rpe enzyme causes changes in the pentose-phosphate levels which alter the regulation of (a) metabolic enzyme(s) and thereby cause growth suppression and that the severity of growth suppression is related to the internal concentration of pentose-phosphates. Target enzymes for negative regulation may be located in the early parts of the Embden-Meyerhof-Parnas pathway and of the Entner-Doudoroff pathway and/or of carbohydrate transport systems feeding sugars into these sections of central metabolic pathways.