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An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.