The O9-specific mannan of E. coli was synthesized in vitro from GDP-14C mannose by membranes which were obtained from a phosphomannose isomerase-less mutant of E. coli O9:K29-:H-. Subsequent treatment of the membranes with dil. acid liberated a neutral product, whereas with aq. PhOH a charged product was obtained. Chromatog. on DEAE-cellulose, incubation with alk. phosphatase, and microdetn. showed that the charged mannan was substituted with 1 phosphate per chain. The neutral 14C-labeled product of the incubation in vitro was reduced with Na boro hydride-3H. After total acid hydrolysis, the radioactive material was chromatographed on paper in the presence of borate. Glucitol-3H, but no mannitol-3H,14C was present. When the neutral product, which was obtained after incubation of 14C-prelabeled membranes with nonradioactive GDP-mannose, was hydrolyzed with and without prior redn. with nonradioactive NaBH4, in subsequent paper chromatog., glucitol-14C or glucose-14C was found. The glucose was also converted enzymically to gluconic acid, which was identified by paper electrophoresis. Apparently, in the neutral O9-specific mannan, glucose is at the reducing end and the mannan chain grows at the nonreducing end. This is discussed with respect to the overall mechanism of the biosynthesis of the O9 antigen.