Laboratory-reared beagles were vaccinated with a placebo or a bacterin comprised of Borrelia burgdorferi S-1-10 and ospA-negative/ospB-negative B. burgdorferi 50772 and challenged after 1 year with B. burgdorferi-infected Ixodes scapularis ticks. For the placebo recipients, spirochetes were recovered from 9 (60%) skin biopsy specimens collected after 1 month, and the organisms persisted in the skin thereafter. Ten (67%) dogs also developed joint infection (3 dogs), lameness or synovitis (7 dogs), or B. burgdorferi-specific antibodies (8 dogs). For the vaccine recipients, spirochetes were recovered from 6 (40%) skin biopsy specimens collected after 1 month. However, subsequent biopsy specimens were negative, and the dogs failed to develop joint infection (P = 0.224), lameness/synovitis (P = 0.006), or Lyme disease-specific antibody responses (P = 0.002). The bacterin provided a high level of protection for 1 year after immunization, and the addition of the OspC-producing B. burgdorferi 50772 provided enhanced protection.