The ring-tailed lemur (Lemur catta) is an endangered species found in southwestern Madagascar, and understanding infectious disease susceptibility is an essential step towards the preservation of wild and captive lemur populations. Lemurs are primates that are widely dispersed throughout the island of Madagascar and may serve as hosts or reservoirs for zoonotic infections. The aim of this study was to determine the prevalence of antibodies to West Nile virus (WNV), simian immunodeficiency virus (SIV), and herpes simplex virus type 1 (HSV-1) in a population of free-ranging ring-tailed lemur from the Beza Mahafaly Special Reserve, Madagascar. Samples were collected from 50 animals during field capture studies in June and July 2004 and assayed for presence of viral antibodies during the 12 mo following collection. Forty-seven of the 50 lemurs sampled had antibodies against WNV detectable by epitope-blocking enzyme-linked immuno-sorbent assay (ELISA). In addition, 50 of 50 samples had titers against WNV ranging from 80 to ≥ 1,280 using plaque reduction neutralization test (PRNT₉₀). Ten lemurs had antibodies against lentiviral antigens as determined by Western blot analysis. None of the lemurs had antibodies against HSV-1 using ELISA.