In the early 1980s, Ixodes spp. ticks were implicated as the key North American vectors of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) (Spirocheatales: Spirochaetaceae), the etiological agent of Lyme disease. Concurrently, other human-biting tick species were investigated as potential B. burgdorferi vectors. Rashes thought to be erythema migrans were observed in patients bitten by Amblyomma americanum (L.) (Acari: Ixodidae) ticks, and spirochetes were visualized in a small percentage of A. americanum using fluorescent antibody staining methods, sparking interest in this species as a candidate vector of B. burgdorferi. Using molecular methods, the spirochetes were subsequently described as Borrelia lonestari sp. nov. (Spirocheatales: Spirochaetaceae), a transovarially transmitted relapsing fever Borrelia of uncertain clinical significance. In total, 54 surveys from more than 35 research groups, involving more than 52,000 ticks, have revealed a low prevalence of B. lonestari, and scarce B. burgdorferi, in A. americanum. In Lyme disease-endemic areas, A. americanum commonly feeds on B. burgdorferi-infected hosts; the extremely low prevalence of B. burgdorferi in this tick results from a saliva barrier to acquiring infection from infected hosts. At least nine transmission experiments involving B. burgdorferi in A. americanum have failed to demonstrate vector competency. Advancements in molecular analysis strongly suggest that initial reports of B. burgdorferi in A. americanum across many states were misidentified B. lonestari, or DNA contamination, yet the early reports continue to be cited without regard to the later clarifying studies. In this article, the surveillance and vector competency studies of B. burgdorferi in A. americanum are reviewed, and we conclude that A. americanum is not a vector of B. burgdorferi.