For the microbiological diagnosis of Lyme borreliosis, antibody detection methods are mainly used. Serological diagnosis should follow the principle of a rational stepwise diagnosis (see English Internet Version (http://www.dghm.org/red/index.html?cname=MIQ) of MiQ Lyme borreliosis). A screening assay (a sensitive ELISA differentiating IgM and IgG) is recommended as the first step. When the ELISA is reactive, it is followed by immunoblot (IgM, IgG) (second step). The reactive diagnostic bands should be clearly identified; this is easy if recombinant antigens are used. Identification of diagnostic bands is difficult in the conventional blot and should be done by monoclonal antibodies. Progress has been made in the sensitivity of the recombinant blot using additional antigens as p58 and homologues of Osp17. In neuroborreliosis the determination of the CSF/serum index is indicated (investigation of paired serum and CSF from the same day). Detection of the etiological agent by culture or PCR should be performed only in specialised laboratories and is confined to specific indications. Recommended specimens are skin, other biopsies, CSF and synovial fluid. The best results are obtained from biopsies (culture and PCR) and synovial fluid (PCR).