To the Editor: West Nile Virus (WNV) is rapidly spreading in the Western Hemisphere (1). We report the first evidence for WNV transmission in South America. WNV is serologically related to the Japanese encephalitis complex of flaviviruses (Flaviviridae), which includes Saint Louis encephalitis virus (SLEV) (in North and South America), Japanese encephalitis virus (Asia), and Murray Valley encephalitis virus (Australia) (2). Because of antigenic cross-reactivity within this complex, WNV serologic diagnosis requires highly specific assays, such as the plaque-reduction neutralization test (PRNT) (3). We used PRNT to evaluate serum collected from 130 healthy equines (horses and donkeys) in Colombia, where WNV had not been previously reported. These equines were sampled between September 15 and October 29, 2004, in the northern departments of Cordoba and Sucre in the Caribbean region of Colombia. Samples were heat-inactivated and titrated by PRNT for antibodies to WNV, SLEV, and 3 other South American flaviviruses: Rocio, Ilheus, and Bussuquara. Twelve specimens (9%) from 10 different premises tested positive for WNV (Table). None of these animals had been vaccinated against WNV or had traveled outside of the region. An equine immunoglobulin (Ig) M-capture enzyme-linked immunosorbent assay (ELISA) that used WNV antigen detected anti-WNV IgM in 2 of the 12 specimens, which indicated that some of these infections were relatively recent (probably within 3 months of sampling). The positive findings in both Cordoba and Sucre were corroborated by a WNV-specific blocking ELISA (4). Numerous other samples exhibited flavivirus reactivity in the neutralization and blocking ELISA assays, mostly because of SLEV. Complete test results from these horses, as well as from Colombian cattle and chickens, will be presented elsewhere. Table PRNT90 antibody titers to WNV and other South American flaviviruses for Colombian equine sera*† These serologic data should be considered indirect evidence of WNV activity in Colombia. We encourage Colombian human and animal health authorities to enhance surveillance for human, equine, and avian disease attributable to WNV. Efforts are needed to isolate the virus or detect specific viral RNA to confirm this finding and to identify vectors and vertebrate hosts involved in WNV transmission in Colombia.