Information on the biosynthesis of the D-arabinans of the cell wall of Mycobacterium tuberculosis is rapidly emerging, with the promise of new targets for drug development against tuberculosis. Accordingly, arabinosyl transferase assays were developed utilizing synthesized [1-14C]-beta-D-arabinofuranosyl-1-monophosphoryldecaprenol as donor and a variety of O- and S-alkyl arabinosides as acceptors. These were: alpha-D-Araf-(1-->5)-alpha-D-Araf-O- and -S-alkyl diarabinosides and alpha-D-Araf-(1-->5)-alpha-D-Araf-(1-->5)-alpha-D-Araf -O- and -S-alkyl triarabinosides. Whereas the O- and S-alkyl monosaccharide acceptors were inactive, the O- and S-alkyl disaccharide and the O- and S-alkyl trisaccharide acceptors (<C12) possessed considerable acceptor activity, and the trisaccharide acceptors were more potent than the corresponding disaccharides. The O-alkyl disaccharide acceptors with a C8 alkyl chain were more active than those containing the C6 or C10 analogs. Chemical analysis of the enzymatically synthesized products of the reactions demonstrated that beta-D-arabinofuranosyl-1-monophosphoryldecaprenol was an effective donor for two of the three potential arabinosyl transferases: beta-D-arabinofuranosyl-1-monophosphoryldecaprenol: arabinan alpha(1-->5) arabinosyl transferase and beta-D-arabinofuranosyl-1-monophosphoryldecaprenol: arabinan beta(1-->2) arabinosyl transferase. The beta(1-->2) arabinosyl transferase activity was more in evidence in the presence of the O-alkyl disaccharide acceptor, whereas both transferases were about equivalent in the presence of the S-alkyl trisaccharide acceptor. The tuberculosis drug, ethambutol, a known mycobacterial arabinosyl transferase inhibitor, was inactive within these arabinosyl transferase/acceptor based assay systems, supporting other evidence that a third activity, responsible for the formation of alpha1-->3 linkage, is the drug target.