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West Nile virus (WNV) was first detected in New Mexico in 2002, with the first human cases appearing in 2003. Since that time it has become endemic in the region, and as of year-end 2005, 330 New Mexicans had been diagnosed with West Nile Fever or the more severe neuroinvasive disease as reported by the New Mexico Department of Health. An ongoing study at the University of New Mexico has collected interview and physical exam data for these individuals as well as collecting cerebrospinal fluid (CSF) and serum from their period of acute and convalescent illness. While all of these samples have been tested to determine WNV IgM seropositivity, none of them have been characterized by the use of nucleic acid amplification test (NAT). The purpose of this study is to characterize this sample set using Real-Time reverse transcriptase polymerase chainreaction (RT-PCR), an extremely sensitive NAT. The serum and CSF archived collection at UNM represents one of the most comprehensive and best-characterized sample sets available in the United States. A total of 115 samples, 111 serum and 4 CSF, were analyzed. None of the 115 samples had detectable West Nile nucleic acid.