Biosynthesis of the cell envelope in mycobacteria is largely unknown; however, several antituberculosis drugs apparently interfere with this process. Recently, we described a lipid intermediate for the biosynthesis of the cell wall arabinogalactan/arabinomannan of Mycobacterium smegmatis: beta-D-arabinofuranosyl-1-monophosphodecaprenol (Wolucka, B. A., McNeil, M. R., de Hoffmann, E., Chojnacki, T., and Brennan, P. J. (1994) J. Biol. Chem. 269, 23328-23335). In the present work, by means of gas chromatography-mass spectrometry, fast atom bombardment tandem mass spectrometry, and proton NMR, the major pentose-containing component of the polyprenyl-P-sugar family from M. smegmatis was characterized as beta-D-ribosyl-1-monophosphodecaprenol (decaprenyl-P-ribose). Additionally, the structure of a minor arabinose-containing compound, beta-D-arabinosyl-1-monophosphooctahydroheptaprenol, could be deduced. In vivo labeling experiments with [14C]glucose demonstrated unequivocally that decaprenyl-P-ribose is actively synthesized in Mycobacterium tuberculosis H37Ra and Mycobacterium avium serovar 4. It is proposed that decaprenyl-P-ribose could be a precursor for the biosynthesis of either some unknown ribose-containing cell envelope polymers of mycobacteria or the arabinan part of the cell wall arabinogalactan/arabinomannan due to the presence of a 2'-epimerase activity at some late stages of the arabinogalactan/arabinomannan biosynthesis.