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Notes: Abstract Specific high affinity binding of the cage convulsant t-[35S] butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S] TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22°C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4°C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD= 50 nM, Bmax= 250 fmol/mg protein in housefly; KD= 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1=8 × 106M-1 min-1), but dissociation was described better by two rate constants (k-1, = 0.28 min-1 and 0.042 min-1; calculated Kd values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50= 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 μM). Stereospecific inhibition by cypermethrin isomers agreed partially with biological activity, suggesting the possible role of this site in some toxicological actions of pyrethroids, although these compounds have more potent effects on voltage-regulated sodium channels. [35S]TBPS binding sites in crayfish muscle appear identical to γ-aminobutyric acid (GABA) receptor-chloride channels labeled by radiolabeled picrotoxin in this tissue. The sites in both invertebrate tissues examined are similar to those in mammalian brain and appear to be the target of important drugs.