Whole-blood samples were obtained from 214 white-tailed deer (Odocoileus virginianus) representing 44 sites in Connecticut (USA) during 1992, 1993, 1996, 1999, and 2000 through 2006. Sera were analyzed for total antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, the respective causative agents of Lyme borreliosis and human granulocytic anaplasmosis. Deer sera contained antibodies to both bacteria during different seasons and throughout the 11-yr study. Of the 224 sera tested, 141 (63%) contained antibodies to whole-cell B. burgdorferi in a polyvalent enzyme-linked immunosorbent assay, whereas 124 (55%) were positive to whole-cell A. phagocytophilum by indirect fluorescent antibody staining. Use of highly specific recombinant antigens (VlsE of B. burgdorferi and protein 44 of A. phagocytophilum) provided strong confirmatory results of past or current infections. There was coexistence of antibodies to whole-cell or recombinant antigens of both agents in 72 (32%) sera. Analyses of 18 sera from eight deer that were marked, released, and recaptured, showed minimal changes in antibody titer over sampling time intervals ranging from 17 days to 5.1 yr. Relatively high antibody prevalences for both bacterial agents in different seasons and years reaffirm that there are well-established foci for both tick-borne infections and probably reflect frequent exposure of deer to infected Ixodes scapularis ticks. November and December is a suitable period to obtain blood samples from deer to conduct serosurveillance for both bacteria.