Streptococcus agalactiae is a major human and animal pathogen, most notable as a cause of life-threatening disease in neonates. S. agalactiae is also called the Group B Streptococcus in reference to the diagnostically significant Lancefield Group B typing antigen. Although the structure of this complex carbohydrate antigen has been solved, little is known of its biosynthesis beyond the identification of a relevant locus in sequenced S. agalactiae genomes. Analysis of the sugar linkages present in the Group B carbohydrate (GBC) structure has allowed us to deduce the minimum enzymology required to complete its biosynthesis. Most of the enzymes required to complete this biosynthesis can be identified within the putative biosynthetic locus. Surprisingly, however, three crucial N-acetylglucosamine transferases and enzymes required for activated precursor synthesis are not apparently located in this locus. A model for GBC biosynthesis wherein the complete polymer is assembled at the cytoplasmic face of the plasma membrane before translocation to the cell surface is proposed. These analyses also suggest that GBC is the major teichoic acid-like polymer in the cell wall of S. agalactiae, whereas lipoteichoic acid is the dominant poly(glycerophosphate) antigen. Genomic analysis has allowed us to predict the pathway leading to the biosynthesis of GBC of S. agalactiae.