We describe the first documented field transmission of West Nile (WN) virus by a North American mosquito. WN was first detected in northern Florida in 2001. An intensive mosquito trapping and surveillance program was conducted in this region for four nights to assess mosquito transmission of WN. Four mosquito traps, each with a single sentinel chicken, were placed at five different locations on each of four nights. A total of 11,948 mosquitoes was collected, and 14 mosquito pools were found to contain WN, giving a minimum infection rate between 1.08 and 7.54 per 1,000. Only one of the 80 sentinel chickens seroconverted to WN, demonstrating a single mosquito transmission event during the study and a mosquito transmission rate of between 0.8 and 1 per 1,000. Culex nigripalpus Theobald was responsible for WN transmission to the sentinel chicken, although both Cx. nigripalpus and Culex quinquefasciatus Say were found infected with WN. Mosquito transmission rates are reported in this study for the first time for a WN outbreak. This information is essential to determine risk of human and animal infection.