The Cry1Ab toxin from Bacillus thuringiensis is expressed by 80% of the commercially available transgenic maize, Zea mays L. that targets the European corn borer, Ostrinia nubilalis (Hübner). The resistance management plan for O. nubilalis is based on the high dose refuge strategy and monitoring of field populations to detect resistance. A collection from Kandiyohi County, Minnesota exhibited significantly lower mortality at the diagnostic concentration developed for O. nubilalis. We used bioassays and biochemical studies to characterize resistance in insects from this field collection. The resistant strain developed high levels of resistance to Cry1Ab and variable levels of cross resistance to other Cry toxins. The inheritance of Cry1Ab resistance in this colony is autosomal, recessive and conferred by three to five genes. Cry1Ab binding to brush border membrane vesicles (BBMV) of susceptible and resistant larvae was compared using surface plasmon resonance and ligand-toxin immunoblot analysis to test the hypothesis that resistance involves reduction of toxin binding to BBMV. We also examined proteolytic degradation of Cry1Ab as a candidate mechanism of resistance.