Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loophelix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors. structure modeling | insecticide action | metamorphosis | Tribolium | Drosophila J uvenile hormone (JH) prevents adult transition (metamorphosis) of insect larvae until they have attained an appropriate stage (1, 2), and it typically stimulates oogenesis in adult females (3). How JH achieves its function remains unclear, mainly because a JH receptor has long eluded identification (4). The lipophilic nature of the sesquiterpene JH suggests an intracellular receptor, yet none of the known insect nuclear hormone receptors have been linked with the biological function of JH. A screen for Drosophila mutants resistant to methoprene (5), a JH mimic and a widely used insecticide (6), uncovered the Methoprene-tolerant (Met) protein containing a basic helix-loop-helix (bHLH) motif followed by two Per-Arnt-Sim (PAS) domains (7). Recombinant Drosophila Met was shown to bind JH at physiological (nanomolar) concentrations and to mediate a weak JH-and methoprene-dependent transcriptional activation in vitro (8). However, Met-null mutant flies were viable and fertile (5), leaving the notion that Met is a putative JH receptor unsupported with an anticipated developmental phenotype. Latest reports show that, in Drosophila, Met might functionally overlap with its paralog, encoded by the germ cell-expressed (gce) gene. Gce can increase sensitivity of Met-null mutants to methoprene (9), and only simultaneous loss of both Met and Gce is lethal (10). However, the actual mode of interaction between JH/methoprene and Met or Gce still remains unclear.Knockdown of the single Met gene in the flour beetle Tribolium castaneum induced beetle larvae to pupate before reaching their final instar (11), producing a precocious metamorphosis phenotype similar to that caused by loss of JH itself (12).Conversely, removal of Met precluded inhibition of adult ...