Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8’,11’,14’-pentadecatriene]-p-benzoquinone), and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity which utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by SAM-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions have not previously been determined. Analyses performed by GC-MS with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3’ position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An EST data set comprised of 5,468 sequences selected at random from a S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real-time RT-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the EST data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3) which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.