Deuterium-labelled fatty acid putative precursors of the 16-carbon aldehydic sex pheromones of Manduca sexta (L.) were applied to the sex pheromone glands of females. After 24 h in vivo incubation, and injection of pheromone biosynthesis activating neuropeptide into the abdomens of treated females, glands were excised, extracted, and the extracts analyzed by gas chromatography-mass spectrometry. Incubation of glands with [16, 16, 16- 2H 3]hexadecanoic acid resulted in the label being incorporated into seven of the eight 16-carbon aldehydes found in the gland, including monoenes, dienes, and trienes. Similarly, incubation with [16, 16, 16- 2H 3](Z)-11-hexadecenoic acid led to incorporation of the label into ( Z)-11-hexadecenal and both conjugated diene and triene aldehydes. However, incubation with [13, 13, 14, 14, 15, 15, 16, 16, 16- 2H 9]( Z)-11-hexadecenoic acid resulted in incorporation of the label into only ( Z)-11-hexadecenal, ( E, Z)- and ( E, E)-10, 12-hexadecadienal, indicating interference of the deuterium isotope in the desaturation process leading to the conjugated triene system. Incubation with the similarly labelled ( E)-11-hexadecenoic acids gave labelled ( E)-11-hexadecenal in both cases, but none of the other unsaturated pheromone aldehydes. The results indicate that fatty acids are the precursors of the aldehydic pheromones in this species and that hexadecanoate is the precursor of the 16-carbon monoenes, dienes, and trienes. The conjugated dienes and trienes are formed via desaturation/isomerization of ( Z)-11-hexadecenoate.