Borrelia burgdorferi is the causative agent of Lyme borreliosis, a multisystem disorder, which can mimic numerous immune disorders and inflammatory diseases. Laboratory diagnosis of Borrelia infection relies on immunodiagnostic assays, which, however, are hampered by unsatisfactory specificity. The Western blot technique has been employed to analyze the humoral immune response in Lyme borreliosis and is used as a serodiagnostic confirmation test. The most important immunodominant proteins of Borrelia burgdorferi are the 94 kDa, 60 kDa, 41 kDa (flagellin), 34 kDa (Osp B), 31 kDa (Osp A), 30 kDa, 21 kDa (Osp C), and 17/18 kDa proteins. Whereas the 60 kDa, 41 kDa, and 34 kDa constituents reveal a marked cross-antigenicity with other spirochetes and even more distantly related bacteria, antibodies against the 94 kDa, 31 kDa and 21 kDa proteins are largely species-specific. The early immune response in Lyme borreliosis is triggered mainly by the flagellin. In the later stage a wide range of immunogenic proteins is involved, with the 94 kDa antigen being the best marker for late immune response. If the Western blot is used for diagnostic purposes the differences between early and late-stage immunogenicity of Borrelia proteins must be taken into account. Interpretation criteria for blot positivity in early-stage borreliosis are primarily based on the presence of the 21 kDa band and the semiquantitatively recorded intensity of the 41 kDa band. In the diagnosis of late-stage infection, blot positivity relies on the presence of the 94 kDa, 39 kDa, 31 kDa, 30 kDa and 21 kDa bands.