An enzyme immunoassay (EIA, ELISA) using microwells coated with a flagellin-enriched fraction of B. burgdorferi and absorbent-containing sample diluent for the quantitative determination of Lyme disease (LD) IgG and IgM antibodies in human serum samples was described. This LD EIA required three 15-minute incubations at room temperature, followed by a 1-step normalization of photometer readings to EIA units (EU/ml). Compared with tests using the whole bacterial extract as antigens and a sample diluent containing 6% BSA, this new LD EIA revealed lower values for 20 syphilis (SS) and 21 normal serum samples (NS) but about the same for 21 Lyme disease (LD) samples, allowing lower cut-off points which would place almost all these SS and NS samples below while almost all LD samples above the positive cut-off point. The LD EIA results of larger numbers (67 to 291) of mixed samples correlated with results of four reference EIA. However, the LD EIA gave lower (2 to 4 fold) reactivities (index values) with SS and NS samples but higher values with positive serum samples than reference EIA. Thus, this LD EIA showed improvements in both specificity and sensitivity over other tests compared.