Laboratory diagnosis of Lyme borreliosis is performed by direct detection of Borrelia burgdorferi in body fluids and tissue samples. This can be achieved by cultivation of the organisms, staining techniques, or demonstration of parts of the genome. Although the best aetiologic proof in case of positivity, these methods cannot yet serve as routine techniques: they are too time consuming and expensive. Currently, the usual method for establishing the diagnosis of Lyme borreliosis is serologic testing (indirect detection). Immunofluorescence, hemagglutination, ELISA tests with whole cell antigen should be considered as screening methods. Assays with selected fractions of B. burgdorferi antigens or tests using selected recombinant antigens should be considered as more specific. Immunoblotting (Western blotting) may be considered as a confirmatory test. However, the interpretation of test results requires an experienced investigator. Laboratory diagnosis of B. burgdorferi infections of the central nervous systems (CNS) is the most highly developed method. Demonstration of intrathecally produced specific antibodies, and, moreover, demonstration of specific oligoclonal bands may very well prove the actual infection of the CNS and/or the nerve roots. Seroepidemiological investigations identify neurological manifestations as the most frequent ones among European cases of Lyme borreliosis. The true incidence and prevalence of Lyme borreliosis, however, cannot be determined with current diagnostic methods and must await the development of methods to identify actual infection.