The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator-containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR-binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.