To evaluate the possible importance of antigenic heterogeneity in the serological diagnosis of Lyme borreliosis a study was performed using antigens from various Lyme Borrelia strains. Serum samples from 102 patients with clinical signs of the infection, all living in an endemic area in southern Sweden, were evaluated by four enzyme immuno assays (EIA). The sera were initially tested for the immunoglobulin G response to antigens from a local Borrelia afzelii strain (ACA1). Serum samples from healthy blood donors residing in the same region were used to define seropositivity in the ACAI-EIA. Immunoblotting was performed with the ACAI antigen and the reactive bands were analysed. A serum was defined as positive when at least four of the Borrelia specific polypeptides (OspC, OspA, OspB, p39, p41 [flagellin], p83, p94, 110kDa) were stained. The same sera were then analysed in three other IgG enzyme immunoassays, one based on antigens from Borrelia burgdorferi sensu stricto B31, and another on pooled protein fractions from strains B31 and ACAI. In the third EIA, sera were analysed for antiflagellin reactivity (B, afzelii strain DK-1). An inconstant immune response was demonstrated in the EIAs and the seropositivity varied between 30-47% when low positive values were excluded, and between 38-73% if all values were included. Fifty sera (50/102) met the criteria for a positive immunoblot, but positive immunoblots were detected with both low positive and negative sera independent of antigen used in the EIAs. Antigens of the local B. afzelii strain were found to detect a higher number of seropositive individuals, which suggests that the antibody reactivity to Lyme Borrelia increases when antigens from a strain endemic in a particular geographical region are used. Data from this study suggest that EIA alone seems insufficient for the serodiagnosis, and antigenic heterogeneity of Lyme Borrelia spp. influences the performance of serum antibody tests. The reliability of serological assays could be increased when the serum antibodies against antigens of Borrelia spp. predominant in the local geographical region are measured.