Polyprenylphosphate-arabinose (in which the polyprenyl unit is found both as decaprenyl and octahydroheptaprenyl) is a donor of mycobacterial cell wall arabinosyl residues. Because of this important role, its biosynthetic pathway, and that of the related lipid, polyprenylphosphate-D-ribose, was investigated. Surprisingly, phosphoribose pyrophosphate was shown to be a key intermediate on the pathway to both polyprenylphosphate-D-pentoses. Thus, incubation of 5-phospho-D-[14C]ribose pyrophosphate with membranes prepared from Mycobacterium smegmatis resulted in the presence of organic-soluble radioactivity that was shown to be, in part, polyprenylphosphate-[14C]arabinose and polyprenylphosphate-[14C]ribose. Two additional intermediates, polyprenylphosphate-5-phospho[14C]ribose and polyprenylphosphate-5-phospho[14C]arabinose, were identified. Further experiments showed that the mature polyprenylphosphate-ribose is formed from phosphoribose pyrophosphate via a two-step pathway involving a transferase to form polyprenylphosphate-5-phosphoribose and then a phosphatase to form the final polyprenylphosphateribose. Polyprenylphosphate-arabinose is formed by a similar pathway with an additional step being the epimerization at C-2 of the ribosyl residue. This epimerization occurs at either the level of phosphoribose pyrophosphate or at the level of polyprenylphosphate-5-phosphoribose.