The objective of this study was to elucidate the biosynthetic route to 4-methyl-1-nonanol, the female-produced sex pheromone of the yellow mealworm beetle, Tenebrio molitor L. The biosynthetic route to the pheromone was examined by (i) allowing the females to feed on defatted bran coated with a stable isotope-labeled putative precursor ([1- 13C]acetate, [1- 13C]propionate, [1- 13C]pentanoate, [1- 13C]2-methylheptanoic acid, or [ 2H 2]4-methylnonanoic acid); (ii) determining if the precursors were incorporated by analyzing the emitted pheromone by gas chromatography/selected ion monitoring-mass spectroscopy (GC/SIM-MS); (iii) where the pheromone was isotopically-enriched, determining the position of the isotopic label(s) through comparison of the MS fragmentation pattern with that of unlabelled 4-methyl-1-nonanol. Although the incorporation of [1- 13C]acetate into 4-methyl-1-nonanol could not be detected, relatively large proportions of the pheromone were produced from the other precursors tested: 81% from [ 2H 2]4-methylnonanoic acid, 45% from [1- 13C]2-methylheptanoic acid, 16% from [1- 13C]pentanoate, and 35% from [1- 13C]propionate (27% from only one unit, and 7.8% from two units). The results indicate that 4-methyl-1-nonanol is produced through a modification of normal fatty acid biosynthesis: initiation of the pathway with one unit of propionate results in the uneven number of carbons in the chain; incorporation of another unit of propionate during elongation provides the methyl branch; reduction of 4-methylnonanoic acid produces the alcohol pheromone. The elucidation of the biosynthetic pathway of 4-methyl-1-nonanol biosynthesis in the yellow mealworm is the first step towards understanding the biochemistry of sex pheromone production in this species.