Background and purpose: Macrocyclic lactones (MLs) are the most widely used broad-spectrum anthelmintic drugs for the treatment of parasitic nematodes impacting both human and animal health. MLs are known to act as agonist of the nematode glutamate-gated chloride channels (GluCls). However, for many important nematode species, the GluCls subunit composition and pharmacological properties remain largely unknown. In order to get new insights about the GluCl diversity and MLs mode of action, we identified and pharmacologically characterized receptors made of highly conserved GluCl subunits from the model nematode Caenorhabditis elegans, the human filarial nematode Brugia malayi and the horse parasite Parascaris univalens. Experimental approach: AVR-14, GLC-2, GLC3 and GLC-4 are the most conserved GluCl subunits throughout the Nematoda phylum. For each nematode species, we investigated the ability of these subunits to form either homomeric or heteromeric GluCls when expressed in Xenopus laevis oocytes and performed the detailed pharmacological characterization of the functional channels. Key results: Here, a total of 14 GluCls have been functionally reconstituted and heteromers formation was inferred from pharmacological criteria. Importantly, we report that the GLC-2 subunit plays a pivotal role in the composition of heteromeric GluCls in nematodes. In addition, we describe a novel GluCl subtype, made of the GLC-2/GLC-3 subunit combination, for which a high concentration of the anthelmintics ivermectin and moxidectin reversibly potentiate glutamate-induced response. Conclusion and implications: This study brings new insights into the diversity of GluCl subtypes in nematodes and promote novel drug targets for the development of next generation anthelmintic compounds.