Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40_LPS backbone structure is composed of a trisaccharide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an L-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145-161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40_LPS form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40_LPS reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that orf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3 also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the L-serine substitution of GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the L-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with L-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.