In Europe, Lyme borreliosis is caused by Borrelia burgdorferi sensu stricto, B. afzelii, B. garinii and the recently described species B. spielmanii. For the development of diagnostic tools, the heterogeneity of the causative agents must be considered. The serological diagnosis should follow the principle of a two-step procedure: a sensitive enzyme-linked immunosorbent analysis as the first step, followed by immunoblot (IgM and IgG) if reactive. The sensitivity and standardization of immunoblots have been enhanced by the use of recombinant antigens instead of whole cell lysates. Improved sensitivity has resulted from the use of recombinant proteins primarily expressed in vivo (e.g. VlsE) and the combination of homologous proteins from different strains (e.g. DbpA). At present, detection rates for serum antibodies are 20–50% in localized, 70–90% in disseminated early and nearly 100% in late disease. Detection of the borreliae by culture or PCR should be confined to specific indications. The best results are obtained from skin biopsies (50–70% with culture or PCR) and synovial tissue or fluid (50–70% with PCR). Cerebrospinal fluid is positive in only 10–30%. Methods that are not recommended for diagnostic purposes include antigen tests in body fluids, PCR of urine and lymphocyte transformation tests.