Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.