The genes of Yersinia enterocolitica serotype O:3 (YeO3) that determine the synthesis of the O-side-chain of the lipopolysaccharide, the rfb region, were cloned into plasmid pBR322. The O-side-chain of YeO3 was expressed by the clone both in Escherichia coli and Salmonella typhimurium indicating that the entire rfb region was included in the clone. It was shown by restriction mapping, deletion analysis and transposition mutagenesis that about 10.4 kilobase pairs of DNA was essential for the synthesis and expression of the O-side-chain. The correct assembly of the O-side-chain on the cell surface of the clone was confirmed by immunofluorescence microscopy and slide agglutination. Immunoblotting using monoclonal antibody specific for the O-side-chain of YeO3 revealed that the O-side-chain material synthesized by the clone in E. coli was similar to that of YeO3. The clone did not show the in vitro temperature variation in O-side-chain expression characteristic of YeO3. Instead analogous O-side-chain was produced both at 25 degrees C and at 37 degrees C. Using transposon Tn2507, which carries a promotorless chloramphenicol acetyltransferase (CAT) gene, transcriptional fusions with the target DNA were generated. When testing the ability of mutated clones to produce CAT, transcription was shown to occur in a uniform direction throughout the whole rfb region. In colony hybridizations, using the cloned insert as a probe, homologous DNA was detected only in pathogenic Y. enterocolitica serotypes.