Mycobacterial diseases, such as tuberculosis and leprosy, are serious human pathogens, in which the cell wall arabinans of arabinogalactan and lipoarabinomannan are essential components of the bacterial cell wall. The chemical synthesis of the key mycobacterial arabinose donor [1-C-14]-beta-D-arabinofuranosyl-1-monophosphoryldecaprenol 2 is described by an application of the phosphoramidite-phosphite triester methodology to form the beta-arabinofuranosyl, allylic phosphodiester. The synthesis uses a novel tert-butyl dimethylsilyl arabinofuranosyl protection strategy, which allows for a regioselective C-1 acid hydrolysis and final full deprotection with ammonium fluoride under mild conditions. A basic arabinosyl transfer assay was developed to study the incorporation of 2 into the cell wall arabinans of mycobacteria. The incorporation was proportional with respect to both the concentration of membrane protein and the acceptor. The epimeric substrate [1-C-14]-alpha-D-arabinofuranosyl-1-monophosporylde-caprenol was inactive and noninhibitorial in this assay. The antimycobacterial drug ethambutol was found to be active suggesting that its mode of action is as an inhibitor of arabinosyl transfer.