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In the literature, there are case reports suggesting that Borrelia burgdorferi infection may induce autoimmune diseases dependent on antinuclear antibodies (ANA). The present study was undertaken in order to verify this possibility in a prospective manner. The study group comprised 78 consecutive patients (51 women and 27 men, median age 41.5 years) referred to our Department for the serologic diagnosis of Borrelia infection. The patients’ sera were tested for Borrelia-specific IgM and IgG (Recombinant Antigen Enzyme Immunoassays, Biomedica). Antibodies against Borrelia were detected in 31 (39.7 %) persons. 15 persons (19.2 %) had positive IgM, another 15 (19.2 %)–positive IgG, and 1 person (3.2 %)–both IgM and IgG. Frequent positivity of IgM antibodies suggests that persons in the early phase of infection prevailed in the group. Tests for anti-dsDNA, anti-RNP, anti-Sm antibodies, and a screening test for systemic rheumatic diseases (ANA Rheuma Screen) were carried out using Varelisa Enzyme Immunoassays (Pharmacia and Upjohn). The spectrum of autoimmune diseases covered by these tests included SLE, MCTD, Sjogren’s syndrome, scleroderma, polymyositis, and dermatomyositis. ANA were detected in 15 persons (19.2 %): anti-dsDNA in 7 (9.0 %), anti-RNP in 1 (1.3 %), anti-Sm in 2 (2.6 %), and ANA Rheuma Screen was positive in 6 persons (7.7 %). Statistical analysis of differences in the ANA frequency between Borrelia-positive and -negative groups was carried out using Fisher’s exact chi-square test (both without and with gender and age matching). No significant differences were found between the groups. Based on the above results, we conclude that there is no increase in the frequency of antinuclear antibodies in the early phase of Borrelia infection.