Glutamate-gated chloride channels (GluCls) are inhibitory ion channels that are sensitive to the antiparasitic drugs ivermectin (IVM) and moxidectin (MOX). We have transiently transfected COS-7 cells with a subunit of a GluCl (HcGluCla) from the parasitic nematode Haemonchus contortus. This subunit bound [3H]-IVM and [3H]-MOX with K(d) values of 0.11+/-0.021 and 0.18+/-0.02nM, respectively. Displacement analysis revealed that IVM and MOX bind to the same site on HcGluCla and that this site is likely distinct from the glutamate binding site. Glutamate was found to be an allosteric modulator of [3H]-MOX and [3H]-IVM binding and increased the affinity of [3H]-MOX for HcGluCla by more than 50% and that of [3H]-IVM by more than 7-fold. These results point to both similarities and differences in the interactions of IVM and MOX with the GluCl. Aspartate, which is structurally similar to glutamate, had little or no effect on [3H]-IVM and [3H]-MOX binding, suggesting that this ligand does not induce the conformational change necessary to potentiate macrocyclic lactone binding. These results also indicate that it may be possible to enhance the efficacy of macrocyclic lactone anthelmintics by administering these compounds with ligands acting allosterically to enhance their binding.