Deuterium-labeled fatty acids have been used to elucidate the sex pheromone biosynthetic pathway in Spodoptera littoralis. Label from palmitic acid was incorporated during the scotophase into all the pheromone acetates and their corresponding fatty acyl intermediates. (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone blend, is synthesized from palmitic acid via tetradecanoic acid, which, by the action of a specific (E)-11 desaturase and subsequently a (Z)-9 desaturase, is converted into (Z,E)-9,11-tetradecadienoate. By further reduction and acetylation, this compound leads to the dienne acetate. Deuterated precursors applied to the pheromone gland during the photophase were also incorporated into the pheromone. The percentage of labeled (Z,E)-9,11-tetradecadienyl acetate relative to natural compound was significantly higher during the light period. Label incorporation from different intermediates into the pheromone was stimulated by injection of brain-subesophageal ganglion extract during the photophase. The influence of the pheromone biosynthesis-activating neuropeptide on the biosynthetic pathway is discussed.