Toxicity and metabolism of t-permethrin were evaluated in two colonies (UF and ARS) of the eastern subterranean termite, Reticulitermes flavipes (Kollar), collected in Gainesville, FL. The UF colony (LC₅₀ = 1.86 μg per vial) was approximately twofold more tolerant of t-permethrin than the ARS colony (LC₅₀ = 0.89 μg per vial) at the LC50. The synergists piperonyl butoxide and S,S,S-tributylphosphorotrithioate increased t-permethrin toxicity four- and threefold (at the LC₅₀) in the UF and ARS colonies, respectively. Despite these differences in t-permethrin susceptibility, microsomal oxidase activities toward surrogate substrates (aldrin epoxidase, and methoxyresorufin O-demethylase), cytochrome P450 content, and microsomal esterase activity toward α-naphthyl acetate did not differ significantly between the colonies. Moreover, no significant differences in qualitative and quantitative metabolism of [¹⁴C]t-permethrin were observed between the UF and ARS colonies for three enzyme sources (microsomal oxidase, microsomal esterase, and cytosolic esterase). Based on in vitro metabolism assays, the major detoxification route of t-permethrin in the UF and ARS termite colonies appears to be hydrolysis catalyzed by microsomal esterases.