Homogenates of Helicoverpa zea (Boddie), Agrotis ipsilon (Hufnagle), and Spodoptera frugiperda (J. E. Smith) third instars and adults contained S,S,S-tri-n-butyl phosphorotrithioate (DEF)-sensitive enzymes that hydrolyzed trans-cypermethrin and two known esterase substrates, α-naphthyl acetate and β-naphthyl acetate. Except for H. zea with α-naphthyl acetate, larval preparations were more active than those of adults, and no marked sex differences were apparent. The hydrolysis of trans-cypermethrin in noctuid preparations were inhibited by DEF, with pI₅₀ values ranging from 4.5 to 6.7. DEF was a potent inhibitor of the degradation of general carboxylesterase substrates α-naphthyl acetate and β-naphthyl acetate in some cases. Electrophoretic studies confirmed the presence in noctuid gut homogenates of one or more DEF-sensitive esterases that hydrolyzed α-naphthyl acetate and β-naphthyl acetate and that were completely inhibited by dichlorvos.