A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encoded Borrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans, n = 31; neuroborreliosis state II, n = 60; Lyme arthritis, n = 24 and acrodermatitis chronica atrophicans, n = 19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.