The present recommendation for serologic confirmation of Lyme disease (LD) calls for immunoblotting in support of positive or equivocal ELISA. Borrelia burgdorferi releases large quantities of proteins, suggesting that specific antibodies in serum might be trapped in immune complexes (ICs), rendering the antibodies undetectable by standard assays using unmodified serum. Production of ICs requires ongoing antigen production, so persistence of IC might be a marker of ongoing or persisting infection. We developed an immunoglobulin M (IgM) capture assay (EMIBA) measuring IC-derived IgM antibodies and tested it using three well-defined LD populations (from an academic LD referral center, a well-described Centers for Disease Control and Prevention (CDC) serum bank, and a group of erythema migrans patients from whose skin lesions B. burgdorferi was grown) and controls (non-Lyme arthritis inflammatory joint disease, syphilis, multiple sclerosis, and nondisease subjects from a region where LD is endemic, perhaps the most relevant comparison group of all). Previous studies demonstrated that specific antigen-antibody complexes in the sera of patients with LD could be precipitated by polyethylene glycol and could then be disrupted with maintenance of the immunoreactivity of the released antibodies, that specific anti-B. burgdorferi IgM was concentrated in ICs, and that occasionally IgM to specific B. burgdorferi antigens was found in the IC but not in unprocessed serum. EMIBA compared favorably with commercial and CDC flagellin-enhanced enzyme-linked immunosorbent assays and other assays in confirming the diagnosis of LD. EMIBA confirmed early B. burgdorferi infection more accurately than the comparator assays. In addition, EMIBA more accurately differentiated seropositivity in patients with active ongoing infection from seroreactivity persisting long after clinically successful antibiotic therapy; i.e., EMIBA identified seroreactivity indicating a clinical circumstance requiring antibiotic therapy. Thus, EMIBA is a promising new assay for accurate serologic confirmation of early and/or active LD.