Highly specific borreliacidal antibodies are induced by infection with Borrelia burgdorferi, and a borreliacidal antibody test (BAT) may be an accurate laboratory procedure for confirming Lyme disease in clinical practice. To investigate this, 34 Lyme disease sera and 34 sera from patients with other illnesses who had presented to a primary-care facility located in an area of borreliosis endemicity were tested by the BAT and Western blotting (WB). The BAT was more sensitive (79% versus 65%; P = 0.090), especially in cases in which patients had a single erythema migrans lesion (P = 0.021). In addition, the potentially cross-reactive sera were negative by the BAT but WB yielded three (9%) false-positive results. The results from 104 sera from possible Lyme disease patients demonstrated the clinical usefulness of the more sensitive and specific BAT. The BAT was positive for 40 (38%) sera from patients with Lyme disease-related symptoms and appropriate clinical and epidemiological findings. WB confirmed Lyme disease in 30 (75%) of the 40 BAT-positive patients but failed to detect B. burgdorferi infection in 10 BAT-positive patients. WB was also positive for 11 BAT-negative sera, but six (55%) patients had case histories which suggested that the results were false positives. Collectively, the results confirm that the BAT is a sensitive and highly specific test and suggest that widespread use would increase the accuracy of serodiagnostic confirmation of Lyme disease.