The prevalence of West Nile virus (WNV) was determined in mosquitoes between November 2002 and October 2004 in East Baton Rouge Parish, LA. A total of 244,374 female mosquitoes were collected and tested by viral isolation. Additionally, 131,896 female mosquitoes were collected in 2003 and tested by VecTest and 167,175 female mosquitoes were collected in 2004 and tested by reverse transcriptase–polymerase chain reaction (RT-PCR). West Nile virus was isolated by cell culture from 17 (47.2%) out of 36 mosquito species collected over the study period. In 2003, WNV was detected in 9 (33.3%) out of 27 species tested by VecTest. In 2004, 14 (50%) out of the 28 mosquito species tested by RT-PCR were positive for WNV. The species with the greatest number of WNV-positive pools detected by all 3 testing methods was Culex quinquefasciatus. A significantly greater proportion of Cx. salinarius pools collected in light traps placed at a 3-m height were positive for WNV by viral isolation than in pools collected in light traps placed at a 1.5-m height.