There are currently no accepted criteria for positive Western blots in Lyme disease. In a retrospective analysis of 225 case and control subjects, the best discriminatory ability of test criteria was obtained by requiring at least 2 of the 8 most common IgM bands in early disease (18, 21, 28, 37, 41, 45, 58, and 93 kDa) and by requiring at least 5 of the 10 most frequent IgG bands after the first weeks of infection (18, 21, 28, 30, 39, 41, 45, 58, 66, and 93 kDa). When these definitions were tested in a prospective study of all 237 patients seen in a diagnostic Lyme disease clinic during a 1-year period and in 74 patients with erythema migrans or summer flu-like illnesses, the IgM blot in early disease had a sensitivity of 32% and a specificity of 100%; the IgG blot after the first weeks of infection had a sensitivity of 83% and a specificity of 95%. Among patients with indeterminate IgG responses by ELISA, 6 of 9 patients with active Lyme disease had positive blots compared with 2 of 34 patients with other illnesses (P < .001). Thus, Western blotting can be used to increase the specificity of serologic testing in Lyme disease.