By crossed immunoelectrophoresis and Western blotting (immunoblotting), it was shown that Borrelia burgdorferi expresses the 60-kilodalton Common Antigen (CA) that is cross-reactive with an equivalent antigen in a wide range of remotely related bacteria. B. burgdorferi CA is strongly immunogenic. A B. burgdorferi genomic library was constructed by using a plasmid cloning system. Escherichia coli recombinants were screened for expression of immunodominant B. burgdorferi antigens. One of the recombinant clones expressed the 60-kilodalton CA of B. burgdorferi. The DNA region encoding B. burgdorferi CA was localized on a 2.3-kilobase fragment of the plasmid pKH1. CA may have pathogenetic implications in Lyme borreliosis, since the CA of mycobacteria recently has been shown to play a role in the etiology of experimental autoimmune arthritis. The extensive cross-reactivity of this antigen may account for the low diagnostic specificity of the currently used serological tests in Lyme borreliosis.