Two distinct metabolic pathways have been elucidated for the formation of isopentenyl diphosphate and dimethylallyl diphosphate, essential metabolic precursors for isoprenoid biosynthesis: the mevalonate pathway, found ubiquitously in mammals, and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, found in most bacteria. As the MEP pathway is absent from mammals, all MEP pathway enzymes represent effective targets for the development of antibacterial drugs. In this study, we found that a herbicide, ketoclomazone, exhibited antibacterial activity against a pathogenic bacterium, Haemophilus influenzae, with an MIC value of 12.5 μg  ml(-1) and that antibacterial activity was suppressed by adding 1-deoxyxylulose, a free alcohol of 1-deoxy-D-xylulose 5-phosphate (DXP). DXP is an MEP pathway intermediate synthesized from pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) by the action of DXP synthase. Thus, we investigated the enzyme kinetics of DXP synthase of H. influenzae (HiDXS) to elucidate an inhibitory mechanism of ketoclomazone on HiDXS. The dxs gene was cloned from H. influenzae and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. The purified HiDXS was a soluble dimeric 70-kDa protein. Steady-state kinetic constants for HiDXS were calculated, and Lineweaver-Burk plots were consistent with a ping-pong bi bi mechanism. The kinetics of inhibition by ketoclomazone suggested that ketoclomazone binds to an unidentified inhibitor-binding site that differs from both the pyruvate-binding site and the D-GAP-binding site on DXP synthase. These data reveal the inhibitory mechanism of ketoclomazone on DXP synthase.