The cap3B gene, which is involved in the formation of the capsule of Streptococcus pneumoniae type 3, encodes a 49-kD protein that has been identified as a polysaccharide synthase. Escherichia coli cells harboring the recombinant plasmid pTBP3 (cap3B) produced pneumococcal type 3 polysaccharide, as demonstrated by immunological tests. Biochemical and cell fractionation analyses revealed that this polysaccharide had a high molecular mass and was localized in substantial amounts in the periplasmic space of E. coli. Unencapsulated (S-2), laboratory pneumococcal strains synthesized type 3 polysaccharide by transformation with plasmid pLSE3B harboring cap3B. In addition, encapsulated pneumococci of types 1, 2, 5, or 8 transformed with pLSE3B can direct the synthesis of pneumococcal type 3 polysaccharide, leading to the formation of strains that display binary type of capsule.