Technical Abstract: Insecticide resistance among Nebraska western corn rootworm populations has been shown to involve increased hydrolytic metabolism, and esterases with intermediate electrophoretic mobility (group II) have been used as a biochemical marker to monitor resistance frequencies in the field. In this study, resistance-associated group II esterases were partially purified by the combined techniques of differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor and % recovery of approximately100-fold and 10%, respectively. Esterases from the resistant and susceptible populations displayed similar chromatographic behavior, however, the activity peak of group II esterases was consistently larger (at least 4-fold) in resistant populations. Kinetic and inhibition studies suggested that the group II esterases are a B-type esterase with similar Km but higher Vmaxin resistant relative to susceptible populations. A putative group II esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII exists as a monomer with a molecular weight around 66 kDa based on size exclusion chromatography of the native enzyme, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. The combined results of this investigation suggest the underlying biochemical basis of esterase-mediated resistance is the overproduction of resistance-associated group II esterases in the resistant D. v. virgifera population.