Following the 1999 West Nile encephalitis outbreak in New York, guidelines were developed to direct surveillance, prevention, and control efforts in the eastern United States. As recommended in the guidelines, the New York City and New York state departments of health developed comprehensive West Nile virus (WNV) surveillance and control programs, which included collecting overwintering Culex mosquitoes to determine whether WNV might persist throughout the winter and initiate a zoonotic transmission cycle in the spring of 2000. As part of this surveillance effort, adult Culex mosquitoes were collected from structures in New York City during January-February 2000 to determine whether overwintering mosquitoes were infected with WNV. This report summarizes the results of this analysis, which documented WNV RNA in some mosquito pools.