In view of recent studies that the ligand-binding pocket of the Drosophila melanogaster nuclear hormone receptor, ultraspiracle (dUSP), is a necessary component of dUSP-dependent transcriptional activation by methyl epoxyfarnesoate, we have assessed qualitative differences in the effect of farnesoid and dodecanoid compounds on receptor conformation and transcriptional activation. Farnesoids possessing terminal alcohol, aldehyde, acid, ester and/or epoxide moieties induced different changes in the local environment of the ligand-binding pocket, as monitored by the change each induced in the fluorescence of the two tryptophan residues existing in dUSP (that are situated 10 residues apart on the alpha-helix 5 that forms one lining of ligand-binding pocket). Similarly, each compound differed in the extent that it promoted an increase in anisotropy (dimerization state) of the receptor. Dodecanoid derivatives were much weaker in causing such effects. Methyl expoxyfarnesoate (insect juvenile hormone III) exhibited the greatest biological activity to increase transcription of a DR12JHECore reporter construct in transfected Sf9 cells, even though it did not exert the most suppression of USP fluorescence nor exert the greatest increase in USP anisotropy. In a comparison of farnesoid derivatives possessing the three side branches either as all methyl groups (JH III), or one of the side branches as ethyl (JH II), or two of the side branches as ethyl (JH I), the JH III and JH I were more similar to each other in the fluorescence suppression and in vivo morphogenetic activity than either was to JH II, evidencing that dUSP does not sense JH II as a structural 'intermediate' between JH III and JH I. Ligand-binding domains of vertebrate retinoid X receptors respond to agonists by repositioning alpha-helix 12 to the edge of a hydrophobic groove, and there with the groove jointly forms a coactivator binding surface. When alpha-helix 12 in dUSP was mutated to place two signaling tryptophan residues its C-terminus, fluorescence signaling indicated that upon dUSP binding of methyl epoxyfarnesoate, the alpha-helix 12 was repositioned differently than what occurred upon binding of non-JH farnesoids. These leads on alternative ligand-induced conformations that dUSP can adopt provide a foundation for commercial development of synthetic molecules that induce specific dUSP conformations, and for identification of in vivo conditions under which endogenous molecules may exert these conformational outcomes to this receptor.