Results of an ELISA, indirect fluorescent antibody (IFA) test, and immunoblot analysis (western blotting) for antibody to Borrelia burgdorferi in a sample of 216 lactating dairy cows were compared. The microscopic microtitration agglutination test for antibody to 6 serovars of Leptospira interrogans was also performed to evaluate possible cross-reactivity between B burgdorferi and L interrogans. Using western blotting as the standard test against which the ELISA and IFA test were compared, the ELISA had greater sensitivity (50% in summer and 38% in spring) with similar specificity (83 and 82%), compared with the IFA test (sensitivity, 6 and 5%; specificity, 90 and 83%). In addition, seropositivity to B burgdorferi, using the ELISA, was not found to be associated with seropositivity to L interrogans serovars. A matched case-control study evaluating the association between clinical lameness and antibody to B burgdorferi was performed in lactating dairy cows of 17 Minnesota and Wisconsin herds. Sera from case and control cows matched by herd, parity, and stage of lactation were evaluated, using an ELISA for B burgdorferi antibody during 2 seasons. High B burgdorferi antibody values were associated with clinical lameness in dairy cows (P = 0.006 in summer and P = 0.04 in spring).