Vaccination with Borrelia burgdorferi outer surface protein (Osp) A can induce a protective response against Lyme disease and serves as a model to understand the generation of protective immune responses against the spirochete. The innate response to pathogens is activated by specific Toll-like receptors (TLRs) that recognize distinct pathogen-associated molecular patterns. TLR2 is of particular interest for B. burgdorferi research because TLR2 recognizes several pathogen-associated molecular patterns, including lipoproteins. TLR2 may form heterodimers with TLR6 to identify diacylated lipoproteins, while TLR2 works in concert with TLR1 to recognize triacylated lipoproteins such as OspA. We will discuss the role of TLR1/2 in the development of responses to OspA in TLR1-and TLR2-deficient mice, and in selected individuals that received the OspA vaccine. While > 95% of human OspA-based Lyme disease vaccine recipients develop OspA antibodies, a very small group of individuals did not develop detectable humoral responses against OspA. We demonstrated that this hyporesponsiveness to OspA vaccination was associated with decreased cell surface expression of TLR1. Moreover, TLR1- and TLR2-deficient mice did not develop significant levels of OspA antibodies following vaccination with OspA, providing a correlation with human hyporesponsiveness to OspA. These data suggest that defects in the TLR1/2 signaling pathway are associated with an impaired ability to generate antibodies following immunization with OspA lipoprotein.