The performances of recombinant borrelial BBK32 proteins as antigens in the serology of erythema migrans (EM) were evaluated in an ELISA. Serum samples were obtained from 75 patients from different geographic areas where three borrelial species, Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii, cause Lyme borreliosis. Antibodies to variant BBK32 proteins were compared with anti-flagella or with anti-IR(6) peptide antibodies. In IgG ELISA at presentation of EM, 65/75 (87 %) patients had antibodies to one or more variants of BBK32, 29/75 (39 %) had antibodies to flagella and 29/75 (39 %) had antibodies to the VlsE IR(6) peptide antigen. The immunoreactivity against variant BBK32 proteins differed in patients from different geographic regions. The present results suggest that the BBK32 proteins used in combination or in parallel may improve the laboratory diagnosis of EM.