To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype O8. A fragment of Y. enterocolitica O8 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90% homology with HSP60 of Y. enterocolitica O3 and GroEL of Escherichia coli and 59% homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65 (amino acid residues 180 to 188) was not found on Yersinia HSP60. To determine whether Yersinia HSP60 is an immunodominant antigen, the immune responses of Yersinia-infected C57BL/6 mice were analyzed. Yersinia-infected mice evolved a significant serum antibody and splenic T-cell response against Yersinia HSP60. CD4+ alpha beta T-cell clones which were generated from splenic T cells isolated from either Yersinia-infected or Yersinia HSP60-immunized mice, recognized both heat-killed Yersinia serotypes O3 and O8 as well as recombinant Yersinia HSP60 but not heat-killed Yersinia pseudotuberculosis, Salmonella typhimurium, or recombinant HSP65 of Mycobacterium bovis. The adoptive transfer of HSP60-reactive T-cell clones mediated significant protection against a lethal infection with Y. enterocolitica O8. These results indicate that HSP60 of Y. enterocolitica is an immunodominant antigen which is recognized by both antibodies and CD4+ alpha beta T cells. Moreover, this is the first report providing direct evidence that microbial HSP may elicit a protective immune response which is not associated with autoimmunity.