West Nile virus (WNV) is an endemic arboviral pathogen that occurs throughout most of the United States and is typically maintained through a bird-mosquito-bird transmission cycle. The ecological significance of the virus is high due to its ability to infect and cause disease in humans, livestock, and wildlife. West Nile virus infection of many vertebrate species causes signs of viral illness, including encephalitis that may result in mortality. Infection by WNV has recently been detected in captive Alligator mississippiensis (American Alligators) in Georgia and Louisiana, and in both captive and free-ranging alligators in Florida. However, additional surveys for WNV in populations of free-ranging alligators within the southeastern USA have not been conducted. The purpose of this study was to survey free-ranging alligators in south Louisiana for active WNV infection. Blood samples were collected from 93 alligators captured at Rockefeller Wildlife Refuge in Cameron Parish, LA, during May 2006 and were screened for WNV using reverse transcriptase polymerase chain reaction (RT-PCR). All samples (100%) tested negative for WNV, indicating a lack of detectable active infection in these animals. Additional surveys of the occurrence of WNV in alligators throughout the southeastern USA are needed to determine the susceptibility of these reptiles to the virus, effects on the health of infected populations, and the potential role of alligators in the maintenance and transmission of the virus.