Our aim was to evaluate the performance of the commercial Immunetics C6 Lyme ELISA assay as a screening assay for anti-Borrelia burgdorferi antibodies in cerebrospinal fluid (CSF). Sensitivity of C6 ELISA was determined in 28 consecutive patients who were diagnosed with neuroborreliosis and had evidence for intrathecal antibody synthesis on immunoblot analysis. The presence of additional bands in CSF or of bands with a stronger intensity in CSF compared with serum was considered evidence of intrathecal synthesis. All 28 patients tested borderline or positive with C6 ELISA on serum. Of the 28 CSF samples, 17 (61%) and 19 (68%) tested positive with C6 ELISA using a threshold of 0.9 and 0.5 (optical density/cutoff). The C6 Lyme ELISA tested has a low sensitivity for antibody detection in cerebrospinal fluid compared with immunoblot analysis.