The aim of the study was evaluation of specific immune response against Borrelia afzelii in patients with erythema migrans, as a sign of an early stage of Lyme borreliosis. The study was performed in 42 patients, residents of Podlasie province, who were initially evaluated by indirect immunofluorescence assay as a seropositive for Borrelia burgdorferi sensu lato. Antibodies against particular antigens of B. afzelii were demonstrated using a Western-blot technique. Evaluation of results showed the most frequent prevalence of antibodies against 41 kDa antigen in majority of patients, which were predominantly of IgM class in patients with short-lasting lesions, and IgG class in patients with long-lasting lesions. Results of IgM blots were recognized as a positive in 26 patients, and IgG blots in 22 patients. Positive result in at least one class of antibodies was noted in 34 patients (81%), and in both classes in 14 (33%). IgM antibodies against 21 kDa antigen, and IgG antibodies against 43 kDa antigen were two fold more frequent in patients with short-lasting lesions. These results confirm the diagnostic usefulness of determination of antibodies against B. afzelii antigens in Poland. The most important for an early diagnosis can be simultaneous demonstration of IgM antibodies against 21 and 41 kDa antigens.