Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA(PG)) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA(LPS)); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA(PG) and ECA(LPS). To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain "monovalent" anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECA(PG), and this suggested that in Y. enterocolitica O:3 ECA(LPS) only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA(PG) and ECA(LPS) expression were found to be regulated by temperature and repressed at 37°C.