Serum samples collected from 623 white-tailed deer (Odocoileus virginianus) in southern Ontario (Canada) from 1985 to 1989 were tested for antibodies to Borrelia burgdorferi using an indirect fluorescent antibody (IFA) staining method. Samples from 150 of the deer were also tested using an enzyme-linked immunosorbent assay (ELISA). At IFA titers of 1:64 and 1:128 deer with antibodies to B. burgdorferi appeared to be widespread throughout southern Ontario, with an apparent prevalence ranging from 3 to 47%. At IFA titres ≥1:256 and ELISA titres ≥1:160 deer with antibodies to B. burgdorferi were only present on Long Point which is the only known endemic focus of Ixodes scapularis, the primary vector for B. burgdorferi, in southern Ontario. At these titres the apparent prevalence of antibodies to B. burgdorferi on Long Point was only 5 to 7%, even though the mean intensity of infestation of adult I. scapularis on deer was >180, and 60% of the adult ticks are infected with B. burgdorferi. Based on these results, white-tailed deer do not appear to be a good sentinel species for the distribution of B. burgdorferi.