We have isolated, cloned, and expressed a male antennae-specific pheromone-degrading enzyme (PDE) [Antheraea polyphemus PDE (ApolPDE), formerly known as Sensillar Esterase] from the wild silkmoth, A. polyphemus, which seems essential for the rapid inactivation of pheromone during flight. The onset of enzymatic activity was detected at day 13 of the pupal stage with a peak at day 2 adult stage. De novo sequencing of ApolPDE, isolated from day 2 male antennae by multiple chromatographic steps, led to cDNA cloning. Purified recombinant ApolPDE, expressed by baculovirus, migrated with the same mobility as the native protein on both native polyacrylamide and isoelectric focusing gel electrophoresis. Concentration of ApolPDE (0.5 microM) in the sensillar lymph is approximately 20,000 lower than that of a pheromone-binding protein. Native and recombinant ApolPDE showed comparable kinetic parameters, with turnover number similar to that of carboxypeptidase and substrate specificity slightly lower than that of acetylcholinesterase. The rapid inactivation of pheromone, even faster than previously estimated, is kinetically compatible with the temporal resolution required for sustained odorant-mediated flight in moths.