Lyme disease is caused by spirochetes of the Borrelia burgdofferi complex and has been reported in many temperate parts of the Northern Hemisphere. The B. burgdorferi complex consists of at least five different species and five genotypes with different pathogenicity. Serodiagnosis was achieved by detection of antigens on one-dimensional (1-D) immunoblots. A systematic and comprehensive approach to elucidate antigens has been started here by the combination of two-dimensional electrophoresis (2-DE) immunoblotting with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Antigens in the proteome of B. garinii BITS were analyzed for their reactivity with sera from patients in early stage (erythema migrans) and late manifestations (neuroborreliosis late, arthritis and acrodermatitis chronica athrophicans) of borreliosis. A strategy to handle the enormous amount of data was developed and 65 antigens were detected, of which 20 were identified. These comprise the known antigens from 1-D immunoblots used routinely in serodiagnosis and additionally the two new antigens, GAPDH and the ABC transporter oligopeptide permease. Several disease-stage unique proteins were detected and some of them identified. The genetic variability between B. garinii strains BITS and 20047, B. afzelii, and B. burgdofferi, sensu stricto, seen on the 2-DE patterns underlines the necessity of the search for additional antigens to improve the serodiagnosis and development of vaccines to be used outside of Northern America. A 2-DE database of B. garinii was built up and is available on the World Wide Web (http://www.mpiib-berlin.mpg.de/2D-PAGE).