The interface between plants and the environment is provided for aerial organs by epicuticular waxes that have been extensively studied. By contrast, little is known about the nature, biosynthesis, and role of waxes at the root-rhizosphere interface. Waxes isolated by rapid immersion of Arabidopsis (Arabidopsis thaliana) roots in organic solvents were rich in saturated C18-C22 alkyl esters of p-hydroxycinnamic acids, but also contained significant amounts of both alpha- and beta-isomers of monoacylglycerols with C22 and C24 saturated acyl groups and the corresponding free fatty acids. Production of these compounds in root waxes was positively correlated to the expression of sn-glycerol-3-P acyltransferase5 (GPAT5), a gene encoding an acyltransferase previously shown to be involved in aliphatic suberin synthesis. This suggests a direct metabolic relationship between suberin and some root waxes. Furthermore, when ectopically expressed in Arabidopsis, GPAT5 produced very-long-chain saturated monoacylglycerols and free fatty acids as novel components of cuticular waxes. The crystal morphology of stem waxes was altered and the load of total stem wax compounds was doubled, although the major components typical of the waxes found on wild-type plants decreased. These results strongly suggest that GPAT5 functions in vivo as an acyltransferase to a glycerol-containing acceptor and has access to the same pool of acyl intermediates and/or may be targeted to the same membrane domain as that of wax synthesis in aerial organs.