Using two monoclonal antibodies, we found subtypes among pneumococcal isolates that are typed as serotype 6A by the quellung reaction. The prevalent subtype bound to both monoclonal antibodies and was labeled here 6Aα, whereas the minor subtype bound to only one monoclonal antibody and was labeled 6Aβ. To determine the biochemical nature of the two serologically defined subtypes, we purified capsular polysaccharides (PSs) from the two subtypes and examined their chemical structures with gas-liquid chromatography and mass spectrometry. The study results for 6Aα PS are consistent with the previously published structure of 6A PS, which is →2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate. In contrast, the 6Aβ PS study results show that its repeating unit is →2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→3) ribitol (5→phosphate. We propose to continue referring to 6Aα as serotype 6A but to refer to 6Aβ as serotype 6C. Serotype 6C would thus represent the 91st pneumococcal serotype, with 90 pneumococcal serotypes having previously been recognized. This study also demonstrates that a new serotype may exist within an established and well-characterized serogroup or serotype. Copyright © 2007, American Society for Microbiology. All Rights Reserved.