The stereochemical course of the Delta(13) desaturation involved in the biosynthesis of Thaumetopoea pityocampa sex pheromone was studied using stereotopically labeled and tagged palmitic acids as metabolic probes. In the synthetic pathway, a functionalized acetylene common synthon was used for introducing the four deuterium tags. Further coupling of the tetradeuterated synthon to the appropriated alkynol and a double chemoenzymatic strategy to resolve the alcohol functionality allowed one to obtain the enantiomerically enriched probes used in the mechanistic studies. Mass spectrometric analyses of extracts from tissues cultured with each probe revealed that removal of the C13 and C14 hydrogens in 11-hexaclecynoate and (Z)-11-hexaclecenoate are pro-(R)- and pro-(S)-specific syn-dehydrogenation processes, respectively. This finding constitutes the first example in the literature of an enzymatic (Z)-desaturation exhibiting a substrate-dependent stereochemical course.