The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1→3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors β-D-Galf-(1→5)-β-D-Galf-O-C10:1 and β-D-Galf-(1→6)-β-D-Galf-O-C10:1, with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both β-D-Galf linkages ((1→5) and (1→6)) found within the mycobacterial cell, as well as in an alternating (1→5) and (1→6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfF, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1→5) and (1→6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.