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Elevated cytochrome P450 monooxygenase-based metabolism is associated with carbaryl and methyl parathion resistance in western corn rootworm (Diabrotica virgifera virgifera LeConte) populations in the state of Nebraska. The objectives of this study were to identify potential differences in cytochrome P450 in resistant and susceptible rootworm populations using the model substrate 4-chloro-N-methyl aniline (4-CNMA) and heme staining of microsomal proteins separated by SDS–PAGE. Baseline N-demethylation activity corresponded well with resistance to the N-methyl-substituted insecticide carbaryl among study populations. N-demethylation of 4-CNMA is NADPH dependent and inhibited by the cytochrome P450 inhibitor piperonyl butoxide, which confirms that the reaction is catalyzed by cytochrome P450. Following ingestion of the cytochrome P450 inducer pentamethyl benzene, N-demethylation activity increased significantly in the more susceptible rootworm populations. Hemoprotein staining of microsomal proteins separated by SDS–PAGE identified two groups of potential P450 isoforms in the 45- to 53-kDa range which appear more highly expressed (a) in resistant populations and (b) following PMB induction.