In this study the raising and development of the immune response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme Borreliosis erythema migrans (EM) was investigated. A total of 95 serially collected serum samples were tested by using three different commercial enzyme-linked immunosorbent assays (ELISAs): recomWell Borrelia, Mikrogen, Enzygnost Borreliosis, DADE Behring, and Quick ELISA C6, Immunetics. The sensitivity of Enzygnost Borreliosis IgM (70.5%) was higher than that of recomWell Borrelia IgM (55.7%), whereas recomWell Borrelia IgG was far more sensitive (57.9%) than Enzygnost Borreliosis IgG (36.8%). Quick ELISA C6 Borrelia was 62.1% sensitive. In order to compare specificity values of the three ELISAs, we tested a panel of sera obtained from blood donors (210 samples coming from a non endemic area and 24 samples from an endemic area) and from patients suffering from some of the most common biological conditions possibly resulting in false-positive reactivity in Lyme disease serology (n=40). recomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1% and 98.9%, respectively), followed by Quick ELISA C6 Borrelia (96.7%). Finally, Enzygnost Borreliosis IgG and IgM achieved 90.1% and 92.3% specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by WB.