Borrelia burgdorferi, the agent of Lyme disease, is recognized by Toll-like receptor (TLR) 1 and 2 heterodimers. Microarray analysis of in vivo B. burgdorferi gene expression in murine skin showed that several genes were altered in TLR1/2-deficient animals compared with wild-type mice. For example, expression of bbe21 (a gene involved in B. burgdorferi lp25 plasmid maintenance) and bb0665 (a gene encoding a glycosyl transferase) were higher in TLR1/2-deficient mice than in control animals. In contrast, messenger RNA levels for bb0731 (a spoJ-like gene) and bba74 (a gene encoding a periplasmic protein) were lower in TLR1/2-deficient mice than in wild-type animals. The expression profiles of some of these genes were altered similarly in B. burgdorferi-infected ticks fed on control or TLR1/2-deficient mice. Quantitative reverse-transcription polymerase chain reaction analysis supported the microarray analysis and suggested that spirochete gene expression is altered by the milieu created by specific host TLRs, both in the murine host and in the arthropod vector.