Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution. In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon. The esterases from the cell-free extract of the Bacillus de-esterified DMP. Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4. The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3. A megaplasmid of about 60 kb was detected in this bacterium. The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing. The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium. The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.