We examined the effect of simulated overwintering temperatures on West Nile (WN) virus replication in Culex pipiens L. derived from mosquitoes collected during the autumn 1999 WN epizootic in New York. The WN virus was a strain isolated from a dead crow also collected during this outbreak. Virus was recovered from most mosquitoes held exclusively at 26°C. In contrast, none of the mosquitoes held exclusively at the lower temperatures had detectable infections. When mosquitoes were transferred to 26°C after being held at 10°C for 21–42 d, infection and dissemination rates increased with increased incubation at 26°C. Future studies involving the attempted isolation of WN virus from overwintering mosquitoes may benefit from holding the mosquitoes at 26°C before testing for infectious virus.