The aim of this study was to determine by Western blotting (WB) the prevalence of anti-outer surface protein C (OspC) IgM and IgG antibodies in patients with Lyme borreliosis according to each of the three genospecies of Borrelia burgdorferi sensu lato. Strains of B. burgdorferi sensu stricto (MUL), B. garinii (DK 6), and B. afzelii (DK 26) served as antigen, all of which expressed abundant OspC. We examined sera from 117 patients with untreated early and late Lyme borreliosis, as well as from 100 blood donors and 29 patients with syphilis. WB results were compared with the B. burgdorferi flagellum enzyme-linked immunosorbent assay (ELISA) data. OspC from B. burgdorferi sensu stricto showed the lowest diagnostic sensitivity. OspC from B. garinii and B. afzelii performed almost identically in erythema migrans, with an IgM positive rate of 36% versus 34%, whereas OspC from B. garinii performed best in neuroborreliosis (60% versus 44%). The anti-OspC IgG response was less prominent than the IgM response and was infrequent in the late stages of the disease (0-20%). The benefit of combining the evaluation of anti-OspC responses with all three species was limited. The overall diagnostic sensitivity of WB anti-B. garinii OspC evaluation was, in the early stages of the disease, comparable to the results obtained using the flagellum ELISA. In erythema migrans and neuroborreliosis, the addition of anti-OspC IgM to the flagellum ELISA increased the sensitivity by 15% and 10%, respectively. It can, therefore, be concluded that OspC from B. garinii is a suitable OspC test antigen, and that supplementary use of OspC from other species adds little to the overall diagnostic sensitivity. An ELISA based on B. garinii OspC and native flagella seems currently the most promising concept for a future antibody test in early Lyme borreliosis.