The structures of the polysaccharide part of lipopolysaccharides isolated from eight Escherichia coli K12/Shigella dysenteriae type 1 hybrids have been determined using sugar and methylation analysis plus 1H- and 13C-nuclear magnetic resonance spectroscopy. The hybrids express parts of the S. dysenteriae type 1 O-antigen tetrasaccharide repeating unit because of the presence of pSS3, a plasmid expressing an alpha-galactosyl: lipopolysaccharide transferase and pSS9, a pBR322 plasmid expressing S. dysenteriae type 1 rfb genes. The various classes of hybrids are the result of transposon Tn 1000 insertions in pSS9 inactivating different rfb genes. The following structural elements were found. E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9-6; a class I hybrid); alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class IV hybrids: E. coli K12 (pSS3, pSS9-36); (pSS3, pSS9-107) and (pSS3, pSS9-114); alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class V hybrids: E. coli K12 (pSS3, pSS9-78) and (pSS3, pSS9-111); alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)bet a-D-GlcpNAc(1-->. The structural sequences are identical to those found in the lipopolysaccharide from native S. dysenteriae type 1. In the hybrid strains, the terminal non-reducing GlcNAc residue of the E. coli K12 core is fully substituted by S. dysenteriae type 1 repeating units, or parts thereof.