Benzoxazinoids are major compounds involved in chemical defence in grasses. These toxins are stored in the vacuole as glucosides. Two glucosyltransferases, BX8 and BX9, that catalyse this last step of benzoxazinoid biosynthesis have been isolated via functional cloning. No close relative of these maize genes was found among the known glucosyltransferases. The enzymes display a very high degree of substrate specificity. DIMBOA, the major benzoxazinoid in young maize, is the preferred substrate. Both genes are highly expressed in young maize seedlings, the developmental stage with the highest activity of benzoxazinoid biosynthesis. Bx8 is included in the cluster of DIMBOA biosynthesis genes located on the short arm of chromosome 4. Hence, the gene cluster comprises three different enzymatic functions and a complete set of genes for the biosynthesis of DIBOA glucoside. Bx9 mapped to chromosome 1. Expression of Bx8 and Bx9 in Arabidopsis corroborated the potency of the enzymes in detoxification of their substrates. This capacity might have implications for allelopathic interactions.