Enteropathogenic Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned ¹H and ¹³C NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: →4)[β-D-Galp-(1 → 3)]-β-D-GalpNAc-(1 → 2)-α-D-Manp-(1 → 3)-α-L-Fucp-(1 → 3)-α-D-GalpNAc-(1→, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by ¹H and ¹³C NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-D-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup.