A Cowdria ruminantium genomic DNA library was constructed in the expression vector lambda ZAPII, and an immunoreactive clone, designated lambda Cr9.4, was isolated by screening with serum from a C. ruminantium-infected goat. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kDa respectively. Database searching indicated that the two genes were homologues of groES and groEL, genes encoding a group of heat shock proteins involved in protein processing, export and assembly. Western blotting experiments showed that the recombinant GroEL protein was recognized by sera raised against four isolates of C. ruminantium which originate from South Africa, West Africa and the Caribbean, but not by antisera to the closely related Ehrlichia species (E. ovina, E. [Cytoecetes] ondiri, E. bovis, E. phagocytophila) of African and European ruminants which can be expected to occur in similar geographical areas to C. ruminantium. This suggests that this protein may be useful in development of serodiagnostic tests for C. ruminantium infection which are not subject to cross-reactions with antibodies to Ehrlichia species. The cloning and expression of the GroE operon will also facilitate further study of the roles of the GroE proteins in the immune response to C. ruminantium.