4,8-Dimethyldecanal (4,8-DMD) is the aggregation pheromone produced by male red flour beetles (RFB), Tribolium castaneum. To elucidate the biosynthetic origin of 4,8-DMD, the following studies were performed: (1) effects of juvenile hormone (JH) III, and pathway inhibitors mevastatin, an inhibitor of the mevalonate pathway, and 2-octynoic acid, an inhibitor of the fatty acid pathway, were tested to determine whether 4,8-DMD is derived from the fatty acid pathway or the mevalonate pathway; (2) incorporation of 13C-labeled acetate, propionate, and mevalonolactone into 4,8-DMD was measured to directly determine the biosynthetic origin of 4,8-DMD; and (3) incorporation of deuterium-labeled precursors, including 2-methylbutanoate (C5D), 4-methylhexanoate (C7D), 2,6-dimethyloctanoate (C10D), and 4,8-dimethyldecanoate (C12D) was tested to determine whether 4,8-DMD is biosynthesized in the sequence Ac-Pr-Ac-Pr-Ac (Ac; acetate, Pr; propionate). JH III was topically applied to males at various doses. Inhibitors and isotopically labeled substrates were administered orally by feeding the beetles flour treated with the substrates of interest, after which volatiles were collected from both sexes of RFBs. The amount of 4,8-DMD produced was significantly reduced with increasing doses of JH III. Also, 2-octynoic acid inhibited the production of 4,8-DMD, but mevastatin did not. Exposure of RFBs to [1-(13)C]acetate and [1-(13)C]propionate, but not [2-(13)C]mevalonolactone, resulted in incorporation of the labeled compounds into 4,8-DMD. RFBs fed flour treated with deuterium-labeled C5D, C10D, and C12D, but not C7D, incorporated these compounds into 4,8-DMD. The findings that the production of 4,8-DMD was inhibited by 2-octynoic acid but unaffected by mevastatin, combined with the high incorporation of [1-(13)C]acetate and [1-(13)C]propionate into 4,8-DMD and the incorporation of deuterated precursors, unambiguously demonstrated that 4,8-DMD is of fatty acid rather than terpene biosynthetic origin, and that the biosynthesis of 4,8-DMD proceeds in the sequence Ac-Pr-Ac-Pr-Ac.