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Sera from 936 mammalian mesopredators (Virginia opossums, gray foxes, striped skunks, hooded skunks, raccoons, a bobcat, and a red fox) were collected during 2003 and 2004 in California, Arizona, Texas, Louisiana, Ohio, and Wyoming and screened for flavivirus-specific antibodies by an epitope-blocking enzyme-linked immunosobent assay (blocking ELISA). Serum samples positive for antibodies against flaviviruses were screened for West Nile virus (WNV)– specific antibodies by blocking ELISA and selectively confirmed with plaque-reduction neutralization tests. High prevalence rates were observed in raccoons (45.6%) and striped skunks (62.9%). The high WNV antibody prevalence noted in mesopredators, their peridomestic tendencies, and their overall pervasiveness make these species potentially useful sentinels for monitoring flaviviruses in defined areas.