Thaumetopoea pityocampa pheromone glands contain an unusual Delta(11) acetylenase that produces an alkynoic fatty acid intermediate in the sex pheromone biosynthetic pathway of this species. In this article, we describe the synthesis and use of the deuterated (Z)-11-hexadecenoic acid probes required to decipher the cryptoregiochemistry of this enzyme. The label in the olefinic bonds was introduced by Wittig reaction between the appropriate deuterated reagents. Besides the vinyl deuterium atoms, for reliable GC-MS analyses these compounds bear a tetradeuterium tag, which was introduced by deuteration of an alkyne intermediate in the presence of the Wilkinson catalyst. Pheromone gland metabolization studies of these probes provided experimental evidence that the transformation of (Z)-11-hexadecenoic acid into 11-hexadecynoic acid by the Delta(11) acetylenase takes place by a stepwise mechanism, in which a significant perturbation of the strong vinyl C11-H bond occurs prior to a fast elimination of the vinyl hydrogen at C-12.