Sorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Delta(9)) to hexadecadienoic acid (16:2Delta(9,12)), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Delta(9,12,15)). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone.