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Tree squirrels (Sciurus spp.) have been recently shown to be commonly exposed to West Nile virus (WNV). Many characteristics of WNV infections in tree squirrels are unknown. To better understand WNV associations in fox squirrels (S. niger), we conducted mark-recapture sampling (N = 72) and radio telemetry to study the longitudinal seroprevalence, seroconversions, and ectoparasites of these animals during 2005–2006 in northern Colorado. Five seroconversions were documented during this study. The majority of seroconversions occurred during the late summer/fall months. However, one seroconversion was documented over the time period of February to late March 2005. Fleas (Orchopeas howardi) were tested for WNV RNA using real-time PCR techniques. No WNV RNA positive fleas (N = 33) were detected. In addition, urine samples (N = 17) opportunistically collected from fox squirrels were negative for WNV RNA. Results indicate that seroconversions can be observed in fox squirrels during low WNV transmission years.