Tobacco budworm,Heliothis virescens,larvae were collected from wild velvet leaf in Macon Ridge (LA) where insecticide resistance in cotton was previously reported. The initial resistance levels were 58.0-fold for thiodicarb and 16.0-fold for cypermethrin compared to a susceptible laboratory population. Selection of this Macon Ridge population with thiodicarb on cotton increased resistance for thiodicarb to 172.9-fold and resulted in cross-resistance for cypermethrin to 161.3-fold compared to the susceptible control. Thiodicarb-selected Macon Ridge budworms were also resistant to methyl parathion (7.6-fold), profenofos (59.9-fold), and azinphosmethyl (>38.8-fold). Cytochrome P450 metabolism ofp-nitroanisole was elevated 30.1-, 16.8-, and 18.8-fold in midgut, fat body, and carcass, respectively, of the selected Macon Ridge budworms. The P450 content was also increased. Ester hydrolysis of 1-naphthyl acetate andp-nitrophenyl acetate as well as 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene glutathioneS-transferase activity were elevated approximately 2-fold with some variability among the specific tissues examined. Piperonyl butoxide increased thiodicarb toxicity by 14.8-fold, methyl parathion by 9.3-fold, and cypermethrin by 19.4-fold.S,S,S-Tributylphosphorothioate increased thiodicarb toxicity by 14.5-fold, methyl parathion by 6.6-fold, and profenofos by 7.2-fold. These results suggests that both cytochrome P450 and esterase play an important role in tobacco budworm resistance and cross-resistance between carbamates, organophosphates, and pyrethroids. Acetylthiocholine hydrolysis was 3.4- and 3.5-fold insensitive to paraoxon and methomyl, respectively, in the thiodicarb-selected Macon Ridge strain. Microassays based onp-nitroanisole andp-nitrophenyl acetate metabolism were successfully used to diagnose resistance in field populations of the tobacco budworm in different geographical areas of the U.S.