Serologic evaluation for the diagnosis of Lyme disease has been confounded by several factors, including a high prevalence of clinically normal dogs testing seropositive, persistence of antibodies, and the introduction of vaccines that will induce antibodies detectable by immunofluorescent antibody assay, whole-cell ELISA, and Western blot assay. The utility of a commercially available in-office test kit (SNAP 3Dx, IDEXX Laboratories) for the simultaneous detection of Borrelia burgdorferi and Ehrlichia canis antibodies and Dirofilaria immitis antigen was evaluated for its ability to detect exposure to B. burgdorferi in both vaccinated and unvaccinated dogs from a highly Lyme-endemic area of Connecticut. The test kit is an ELISA that uses a synthetic peptide (C6) that duplicates the sequence of the IR6 region. The in-office C6 ELISA kit was found to be particularly useful in Lyme-endemic areas because it can be used conveniently and reliably in the clinic to determine a dog's infection status regardless of the vaccination history of the animal.