Select pyrethroid binary mixtures (deltamethrin plus S-bioallethrin, β-cyfluthrin, cypermethrin, and fenpropathrin) elicit a more-than-additive response on L-glutamate release from rat brain synaptosomes that is independent of calcium influx. Using a variety of chloride channel antagonists, anthracene-9-carboxylic acid (9-AC), rChlorotoxin (ClTx), 4,4’-dintitrostilbene-2,2’-disulfonic acid (DNDS), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and picrotoxinin (PTX), we have identified two mechanisms by which pyrethroids may enhance L-glutamate release. The results from this study indicate that only ClTx and NPPB, at their EC50s (0.1 μM and 70 μM, respectively), significantly increase L-glutamate release when in the presence of our most potent pyrethroid, deltamethrin, at its EC50 (2 x 10-12 M). When these two antagonists were used in the presence of deltamethrin plus cypermethrin and deltamethrin plus fenpropathrin, a more-than-additive response was elicited at lower concentrations of the binary mixtures. Likewise, NPPB in the presence of the additive binary mixture, deltamethrin plus tefluthrin, first elicited a more-than-additive response at the 1:10 mixture. Since both ClTx and NPPB are inhibitors of voltage-gated chloride channels (ClC-2) and calcium-activated chloride channels, our findings suggest that these channels are potential target sites for certain pyrethroids and likely are important in pyrethroid neurotoxicity.