The type II class of sex pheromones found in moths is composed of polyene hydrocarbons and their epoxides. Analysis of Utetheisa ornatrix females by gas chromatography-mass spectrometry and measurement of responses of male moths by coupled gas chromatography-electroantennogram detection confirmed the presence of large amounts of (Z,Z,Z)-1,3,6,9-heneicosatetraene (1,3,6,9-21:Hy) and smaller amounts of (Z,Z,Z)-3,6,9-heneicosatriene (3,6,9-21:Hy). Both compounds were detected in pheromone glands of newly emerged adults, with low amounts found in the late pupal stage, indicating that sex pheromone biosynthesis started in the late pupal stage. In our population of females (several hundred sampled), approximately 90% produced the tetraene, 1,3,6,9-21:Hy, as the major component, while the other 10% produced only a large amount (1500–2000 ng) of 3,6,9-21:Hy, with no detectable amount of the tetraene. This result could indicate that two distinct populations are present in our original collection site in Florida. Decapitated female moths accumulated 3,6,9-21:Hy and 1,3,6,9-21:Hy compared to the same age normal females, indicating that female moths continuously produce pheromone. A pheromone biosynthesis activating neuropeptide (PBAN)-like neuropeptide did not affect sex pheromone production as indicated by injection of synthetic PBAN and decapitation of U. ornatrix female adults. When the labeled precursor, D4-9,12,15-18:acid, was injected into the early pupal stage, the most abundantly labeled hydrocarbons were 3,6,9-21:Hy and 1,3,6,9-21:Hy in the female adults. This result indicated that 3,6,9-21:Hy could be biosynthesized from linolenic acid through chain elongation and decarboxylation. To determine how 1,3,6,9-21:Hy is produced, D4-3,6,9-21:Hy was injected into pupae and monitored for incorporation of label. No label was incorporated into 1,3,6,9-21:Hy, although a large amount of triene, 3,6,9-21:Hy, was recovered in the pheromone gland. This indicates that U. ornatrix females do not use 3,6,9-21:Hy to produce 1,3,6,9-21:Hy, and the terminal double bond is introduced earlier in the biosynthetic pathway.