A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistance to Bacilus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains ofH. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2-to 4-fold reduction in binding affinity was accompanied by a 4-to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryMA, nor did these proteins compete with the CryLA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.Insect-control proteins from Bacillus thuringiensis ssp. kurstaki are active against a wide range of agronomically important lepidopteran larvae (1, 2). Use of these proteins in optimized microbial strains and genetically improved plants will become increasingly important for insect control (3), particularly as the popularity of many commercial chemical insecticides is declining due to the onset of resistance by target pests (4). Although commercial preparations of B. thuringiensis strains have been used for >25 yr, only recently have insects with reduced susceptibility been identified (5) and obtained in laboratory-selection experiments (6-8). Management strategies are being developed to prevent or delay the onset of insect resistance to assure the long-term efficacy of B. thuringiensis proteins. Biochemical characterization of B. thuringiensis proteins, their mode of action, and mechanisms of increased resistance are critical for the development of appropriate management strategies.The mode of action of B. thuringiensis protein insecticides is complex, as evidenced in a number of reports over the last few years (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Upon ingestion by the insect, the proteins are proteolytically processed (9-14), cross the peritrophic membrane, and bind to high-affinity receptors on the midgut epithelium (15)(16)(17)(18)(19)(20). The membrane-bound B. thuringiensis protein disrupts the membrane integrity by forming a pore, causing an electrolyte imbalance that ultimately kills the insect (21,22). Receptor binding has been analyzed by usingmidgut brush-border vesicles from the gut epithelium from various lepidopteran larvae and for several B. thuringiensis proteins (16,(18)(19)(20)23). High-affinity binding sites were initially identified that directly correlated with the ob...