The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti-WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood-brain barrier (BBB) in WN virus-infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto-occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM-capture-enzyme-linked immunosorbent assay (MAC-ELISA) was used to detect anti-WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P < .02) IgG index compared to the AO site (0.34 +/- 0.04 versus 0.22 +/- 0.04 [mean +/- SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC-ELISA testing. However, the positive to negative antigen ratios were higher (P < .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC-ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.