We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aα and 11Aβ. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11Aα was determined to be identical to 11A. 11Aβ is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11Aα and 11Aβ subtypes. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.