The in vivo ligand-binding function and ligand-binding activity of the Drosophila melanogaster retinoid-X receptor (RXR) ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence-binding assay, farnesoid products in the juvenile hormone (JH) biosynthesis pathway, and their epoxy derivatives, were measured for their affinity constant for ultraspiracle (USP). Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focused for a ring-gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland, methyl farnesoate, was identified possessing these features and exhibited an affinity for ultraspiracle (K(d) = 40 nm) of similar strength to that of RXR for 9-cis retinoic acid. Mutational analysis of amino acid residues with side chains extending into the ligand-binding pocket cavity (and not interacting with secondary receptor structures or extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) showed that the mutation C472A/H475L strongly reduced USP binding to this ring gland product and to JH III, with less effect on other ring-gland farnesoids and little effect on binding by (the unnatural to Drosophila) JH I. Along with the ecdysone receptor, USP is now the second arthropod nuclear hormone receptor for which a secreted product of an endocrine gland that binds the receptor with nanomolar affinity has been identified.