This study shows that the pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of sex pheromone biosynthesis in Thaumetopoea pityocampa. In vitro incubation of pheromone glands with PBAN resulted in a dose-dependent increase in pheromone titer. The formation of labeled pheromone from deuterated 11-hexadecynoic acid was stimulated in pheromone glands upon in vitro incubation with PBAN. However, no differences were found between PBAN-treated glands and controls in the amounts of labeled intermediates. In vivo experiments with deuterated (Z)-11-hexadecenoic acid and 11-hexadecynoic acid showed that 12,13-methylenehexadec-12-enoic acid inhibited both the δ-11 desaturation of (Z)-11-hexadecenoic acid and the Z-13 desaturation of 11-hexadecynoic acid. The amounts of natural pheromone produced by pheromone glands topically treated, in vivo, with 12,13-methylenehexadec-12-enoic acid and further incubated, in vitro, with PBAN were similar to those found in glands that had not been administered the inhibitor. Topical application of (Z)-13-hexadecen-11-yn-l-ol to pheromone glands in vivo resulted in the formation of the corresponding acetate. All these results indicate that PBAN controls pheromone biosynthesis in the processionary moth by regulating a step involved in the transformation of the enyne acyl intermediate into the enynol.