The role of red fox, Vulpes vulpes, and its associated ticks in maintaining Borrelia burgdorferi sensu lato (s.l.) was studied. A total of 1583 ticks were removed from ears of 120 infested animals and were identified as species using a nested PCR targeting the ITS2 and coxI fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga, I. ricinus, and I. hexagonus. In total, ... [Show full abstract] 32.4% of 943 ticks revealed Borrelia DNA and 10 species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii comprised 70% of all infections. The other eight species included B. americana, B. bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. lanei, B. spielmanii, and B. valaisiana. Analysis of tissues from 243 foxes showed that 23.5% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii, the second most prevalent species, followed by B. spielmanii. The predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with foxes. Although red foxes are exposed to a high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.