Paraherquamide was identified recently as a potent anthelmintic agent. In this paper we describe the identification and characterization of a specific, high-affinity paraherquamide binding site in a membrane preparation isolated from the free-living nematode, Caenorhabditis elegans. [3H] Paraherquamide bound specifically to C. elegans membranes with an apparent dissociation constant, Kd, of 263 nM. A series of paraherquamide analogs were examined, and their relative affinity for the paraherquamide binding site correlated with their nematocidal activity. Phenothiazines were the only other class of anthelmintics tested which inhibited specific [3H]paraherquamide binding. These results suggest that the anthelmintic activity of paraherquamide and phenothiazine is mediated via an interaction with a common binding site.