Toll-like receptor 2 (TLR2) plays an important role in the recognition of Borrelia bacteria, the causative agent of Lyme disease, but the existence and importance of additional receptors in this process has been hypothesized. In the present study, we confirmed the role played by TLR2 in the recognition of Borrelia bacteria but also demonstrated a crucial role for the intracellular peptidoglycan receptor NOD2 for sensing the spirochete. Cells from individuals who were homozygous for the loss-of-function mutation 3020insC in the NOD2 gene were defective with respect to cytokine release after stimulation with Borrelia species, and this was confirmed in peritoneal macrophages from mice lacking RICK, the adaptor molecule used by NOD2. In contrast, NOD1 played no major role in the recognition of Borrelia spirochetes. This raises the intriguing possibility that recognition of Borrelia spirochetes is exerted by TLR2 in combination with NOD2 and that both receptors are necessary for an effective induction of cytokines by Borrelia species. The interplay between TLR2 and NOD2 might not only be necessary for the induction of a proper immune response but may also contribute to inflammatory-induced pathology.