The O-polysaccharide from Vibrio cholerae O:5 has been investigated, using NMR spectroscopy as the main method. Fast atom bombardment mass spectrometry (FABMS) studies of fragments obtained on treatment with anhydrous hydrogen fluoride or methanolic hydrogen chloride gave further structural information. Some structural features were also determined by comparison of nuclear Overhauser enhancement (NOE) contacts with calculated H-H distance in different oligosaccharide models. It is concluded that the O-polysaccharide has the following structure, in which D-Qui p NAc4NAc is 2,4-diacetamido-2,4,6-trideoxy-D-glucose, and D-Fuc p 3NX is 3-amino-3,6-dideoxy-D-galactose acylated with a (R,R)-3-hydroxy-3-methyl-5-oxoproline group. [formula: see text]