In this article, we report the first stereochemical study of an enzymatic 1,4-dehydrogenation reaction, namely, the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, involved in the sex pheromone biosynthesis of the moth Spodoptera littoralis. The investigation was carried out using the labeled substrates (R)-[10-(2)H]- and (S)-[10-(2)H]-tridecanoic acids ((R)-2 and (S)-2, respectively) and (R)-[2,2,3,3,13-(2)H(5)]- and (S)-[2,2,3,3,13-(2)H(5)]-tetradecanoic acids ((R)-1 and (S)-1, respectively). Probes (R)-2 and (S)-2 were prepared as described in a previous article.(1) The synthesis of the pentadeuterated chiral substrates (R)-1 and (S)-1 was accomplished by kinetic resolution of the racemic 12-tridecyn-2-ol (6) with immobilized porcine pancreatic lipase. The enantiomerically pure alcohols (R)-6 and (S)-6 were transformed into the final acids (S)-1 and (R)-1, respectively, by a sequence of well-established reactions. The analyses of methanolyzed lipidic extracts from glands incubated separatedly with each individual probe showed that in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, both pro-(R) hydrogen atoms at C-10 and C-13 are removed from the substrate. This is the first example reported of a desaturase with pro-(R)/pro-(R) stereospecificities that gives rise to (E)-double bonds. A mechanistic explanation for the stereochemical outcome of this reaction is advanced.