In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes. Divergently with respect to the edd gene is the gap-1 gene. Expression from P(zwf), P(edd), and P(gap) is modulated by HexR in response to the availability of glucose in the medium. To study the regulatory process in greater detail we purified HexR and showed that it is a monomer in solution. Electrophoretic mobility shift assays and isothermal titration calorimetry assays were done showing that HexR recognizes the P(edd), P(zwf), and P(gap-1) promoters with affinity in the nanomolar range. DNA footprinting assays identified the binding site between +30 and +1 at P(zwf), between +16 and +41 at P(edd), and between -6 and +18 at P(gap-1). Based on DNA sequence alignment of the target sites and isothermal titration calorimetry data, two monomers of HexR bind to a pseudopalindrome with a consensus sequence of 5'-TTGTN(7-8)ACAA-3'. Binding of the Entner-Doudoroff pathway intermediate 2-keto-3-deoxy-6-phosphogluconate to HexR released the repressor from its target operators, whereas other chemicals such as glucose, glucose 6-phosphate, and 6-phosphogluconate did not induce complex dissociation. The phosphorylated effector is likely to be recognized by a sugar isomerase domain located at the C-terminal end of HexR, whereas the helix-turn-helix DNA binding domain of HexR exhibits high similarity to proteins of the RpiR family of regulators.