Abstract Previous studies had suggested that mannosyl-1-phosphosphoryl-undecaprenol was an intermediate in the transfer of mannosyl units from GDP-mannose to mannan. In this report Triton X-100 and EDTA were used as selective inhibitors of the enzymatic synthesis of mannan. As a result of these studies the hypothesis that mannosyl-1-phosphoryl-undecaprenol was an obligatory intermediate in mannan biosynthesis was confirmed. The distribution of radioactive mannosyl units in mannan prepared in vitro was analyzed with the techniques of exhaustive methylation, acetolysis, and enzymatic digestion. Exhaustive methylation revealed that only the nonreducing terminal positions had become labeled as a result of the enzymatic reaction. Mannan formed in vitro was degraded by acetolysis to primarily disaccharides and trisaccharides; in both types of oligosaccharides the radioactivity was concentrated in the nonreducing termini. Enzymatic digestion with α-mannosidases released a large proportion of the radioactivity present in mannan formed enzymatically and thereby confirmed the results of methylation and acetolysis. As a result of exhaustive methylation the mannan was found to contain 1 → 2, 1 → 3, and 1 → 6 mannosidic linkages and the extent of branching in the polysaccharide was low.