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Spirochete of Borrelia burgdorferi sensu lato were isolated in a modified BSK medium from 26 patients of 275 investigated suffering from early and late borreliosis. The isolates were specified by phenotype and genotype analysis using monoclonal antibodies, by immunochemical methods and by including species specific 16S rRNA, OspA and OspC primers and probes in a polymerase chain reaction with hybridization. Borrelia afzelii was found in 9 patients, Borrelia garinii in 16 and B. burgdorferi sensu stricto in one instance. B. afzelii was isolated from 5 biopsies with erythema migrans. 2 with acrodermatitis chronica atrophicans and from blood of 2 patients with arthritis. B. garinii cultures were prepared from 6 cerebrospinal fluids, 3 blood samples, 1 placenta, 1 liver biopsy and 5 skin samples. Unique was B. burgdorferi detected in a bioptic sample of the heart muscle which was positive on immunohistochemical examination. Monoclonal antibodies against OspA and genotype analysis provided evidence that Borrelia garinii isolates from cerebrospinal fluid are close to serotype IV. One CSF isolate was resistant to antibiotics; the tropism of B. garinii to nervous tissue is contemplated.