Dogs were challenged with Borrelia burgdorferi by exposure to ticks, with or without prior protection from infection by recombinant OspA (rOspA) vaccination. Sera from these dogs were tested for their capability to inhibit the growth of B. burgdorferi in vitro. Bacterial growth was detected by a color change in the culture medium, and the optical density was measured with a spectrophotometer in microtiter plates. By growth inhibition, which was complement dependent, the color change was lacking after 5 days of incubation. Over a 1-year study, nonvaccinated dogs infected by exposure to ticks showed high antibody titers to B. burgdorferi by kinetic enzyme-linked immunosorbent assay (KELA). The same sera did not inhibit spirochetal growth or did so only at a low dilution. These results corresponded to the lack of OspA and OspB antibodies seen in Western blots (immunoblots), and these dogs were not protected from infection or disease. In contrast, dogs immunized with rOspA prior to challenge with infected ticks produced high antibody titers, as determined by KELA, but their sera also had high growth-inhibiting antibody titers. Western blot analysis showed a strong band in the 32-kDa region when the sera of these dogs were tested. When adjuvant was administered with rOspA, antibody titers by both KELA and growth inhibition were higher and persisted longer in the immunized dogs. All dogs immunized with rOspA were protected from infection and disease.