Copyright © 2017 American Society for Microbiology. Streptococcus pneumoniae expresses capsular polysaccharides (CPSs) to protect itself from opsonophagocytic killing. The genes responsible for capsules synthesized by the Wzy-dependent mechanism, which accounts for 96 of the 98 known pneumococcal capsule types, are in a chromosomal region known as the cps locus. The nucleotide sequence in this region has been determined for all serotypes. In contrast, not all CPS structures have been defined. The structure of the serotype 35C polysaccharide was recently reported, but the presence of O-acetyltransferase genes in the serotype 35C cps locus suggested that it could be incomplete, as the reported structure contains no O-acetylation. In addition, the genetic distinction of serotype 35C from the closely related serotype 42 was unclear, as their reported cps loci are nearly identical. To clarify these discrepancies, we obtained serotype 35C and 42 clinical and reference isolates and studied their serological and genetic properties, as well as the structures of CPSs purified from reference isolates. We demonstrated that the Oacetyltransferase WciG was functional in serotype 35C but nonfunctional in serotype 42 due to a deletion in wciG. Serotype 35C was O-acetylated at the 5- and 6-positions of 3-β-galactofuranose, as well as the 2-position of 6-β-galactofuranose. However, serotype 42 has only O-acetylation at 3-β-galactofuranose, an observation consistent with its loss of WciG functionality, which is associated with O-acetylation at the 2-position and subsequent reaction with typing antiserum 35a. These findings provide a comprehensive view of the genetic, biochemical structural, and serological bases of serotypes 35C and 42.