Serum samples were obtained from white-footed mice (Peromyscus leucopus) in tick-infested areas of Connecticut during the period 2001 through 2003 and analyzed for antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Emphasis was placed on the evaluations of highly specific recombinant VlsE or protein (p) 44 antigens of B. burgdorferi and A. phagocytophilum, respectively, in a newly developed enzyme-linked immunosorbent assay (ELISA) as well as testing sera with whole-cell antigens by conventional ELISA or indirect fluorescent antibody staining methods. Of the 414 mouse sera analyzed, 310 (75%) had antibodies to whole-cell B. burgdorferi, whereas 157 (38%) were positive to the VlsE antigen. The latter nearly equaled the overall antibody prevalence rate (37%) computed when sera were tested separately with the p44 antigen. Mice were exposed to these pathogens and B. microti (antibody prevalence = 25%) in extreme northern Connecticut as well as the southern coastal areas of the state, thus indicating further geographic expansion of these infections. Fifty-three (13%) sera from widely separated sites had antibodies to all three pathogens. With expression and immunological recognition of VlsE and p44 antigens in P. leucopus, separate incorporation of these fusion proteins in an ELISA was very helpful in confirming past or current infections and in identifying specific foci for B. burgdorferi and A. phagocytophilum.