Previous studies have shown that polymerized [14C]arabinan can be synthesized from polyprenylphosphate-[14C]arabinose by the particulate enzymes of Mycobacterium smegmatis [R.E. Lee, K. Mikusová, P.J. Brennan and G.S Besra (1995) J. Am. Chem. Soc. 117, 11829-11832]. In the present investigation, the [14C]arabinan product was biochemically characterized. Sizing chromatography revealed a molecular weight consistent with that expected from mature arabinan. Digestion of the [14C]arabinan with a mixture of arabinases produced oligo[14C]arabinoside fragments including hexa[14C]arabinoside and tetra[14C]arabinoside which originated from the non-reducing terminal regions of the polymer, and di[14C]arabinoside from the internal regions of the polymer. These arabinoside fragments represent the major known structural motifs that comprise the arabinan segment of arabinogalactan and lipoarabinomannan. The presence of [14C]arabinose in both the internal and external regions of the [14C]arabinan suggests that polyprenylphosphate-arabinose is the major, and perhaps the only, donor of arabinosyl residues in mycobacteria.