To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2^−/− C3H/HeJ mice. All TLR2^−/− mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2^−/− mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2^−/− than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2^−/− mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.