Mosquitoes identified as female Culex (Culex) species, primarily mixtures or uniform batches of Culex pipiens and Culex restuans, were collected daily from gravid traps by 2 mosquito abatement districts (MADs) in Cook County, Illinois. From 2002 through 2004, batches (pools) of mosquitoes were tested by the MADs for West Nile virus (WNV) by using VecTest WNV antigen assays and the same samples were retested, usually within 1-2 wk, for WNV RNA by the TaqMan reverse transcriptase polymerase chain reaction (RT-PCR). There were 952 TaqMan-positive pools out of 3,953 pools over the 3 years, and about one half of that number were VecTest-positive. The difference between the 2 detection assays varied between and within years. The VecTest assays detected about 57% and 69% of the TaqMan RT-PCR-positive pools from Des Plaines Valley MAD and Northwest MAD in 2002, but only about 40% and 46% in 2003, and 36% and 55% in 2004, respectively. Based on a subset of the 2004 data, a linear relationship was found between VecTest detection of WNV and TaqMan cycle threshold between 18 and 28 cycles. A temporal decrease in the difference between the 2 assays was observed in 2003 and 2004, which we conjecture is due, at least partially, to a seasonal decline in the proportion of recently infected mosquitoes. This trend was not observed in 2002 because infection rates indicated a high likelihood of more than 1 infected mosquito per pool at the peak of transmission. Unlike a previous study, the 95% confidence intervals of infection rates based on the 2 detection methods did not always overlap. The highest infection rates occurred in 2002 when mean monthly temperatures were above average.