Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.