The midgut proteinases from a Bacillus thuringiensis-susceptible (IA-S) and four laboratory-selected resistant strains (KS-SC, KS-NE, IA-1, and IA-3) of European corn borer (Ostrinia nubilalis) were characterized using three synthetic substrates, Nα-benzoyl-L-arginine p-nitroanilide (BApNA) for trypsin-like, N-succinyl-ala-ala-pro-phe p-nitroanilide (SAAPFpNA) for chymotrypsin-like, and N-succinyl-ala-ala-pro-leu p-nitroanilide (SAAPLpNA) for elastase-like proteinase activities. The hydrolyzing efficiency of trypsin-like proteinases, determined by Vmax, decreased 35% in the KS-SC resistant strain compared with the susceptible strain. There were no significant differences in the Michaelis constant (Km) among the five strains for the same substrate. When the purified B. thuringiensis Cry1Ab protoxin was used as the substrate, there was a detectable reduction in the hydrolysis of protoxin that was mediated by midgut proteinases from the KS-SC strain compared with the IA-S strain. Thus, the reduced trypsin-like proteinase activity appeared to lead to the reduced activation of the B. thuringiensis protoxins. This may confer or contribute to B. thuringiensis resistance in this strain. However, no significant difference was found in trypsin activity between the IA-S strain and the three other resistant strains (i.e., KS-NE, IA-1, and IA-3) and in chymotrypsin activity among all strains examined. These results suggest that other resistance mechanisms are responsible for the B. thuringiensis resistance in the KS-NE, IA-1, IA-2, and IA-3 strains of European corn borer.