To determine whether rodents that are intensely exposed to the deer tick-transmitted agents of Lyme disease, human granulocytic ehrlichiosis, and human babesiosis are also exposed to deer tick virus (DTV), we assayed serum samples from white-footed mice (Peromyscus leucopus) and meadow voles (Microtus pennsylvanicus) in sites densely infested by deer ticks. To conduct serosurveys, we developed an enzyme-linked immunosorbent assay (ELISA) and Western blot assay by cloning, expressing, and purifying a portion of the DTV envelope glycoprotein (DTV rE) for use as test antigen. Sera from mice and voles trapped in Massachusetts, Rhode Island, and Wisconsin were screened by ELISA for IgG reactive to DTV rE. Samples that were positive or borderline by ELISA were subsequently analyzed by immunoblotting. Samples reactive in both assays were considered to be positive. Three percent of 264 mouse samples collected from sites in Rhode Island, 3.8% of 52 samples from mice trapped in Wisconsin, and 3.9% of 282 samples collected from mice trapped on Nantucket Island, MA were positive. No samples from either Great Island, MA, or voles from any study site were reactive. A reverse transcriptase-polymerase chain reaction yielded molecular evidence of DTV infecting questing adult deer ticks in sites where seroreactive mice were trapped, but not from ticks collected where serologic evidence of virus perpetuation was absent. White-footed mice appear to be exposed to DTV in certain sites where other deer tick-borne agents perpetuate. This virus may be maintained in the same enzootic cycle.