A blocking enzyme-linked immunosorbent assay was used to test 97 serum samples from big brown bats (Eptesicus fuscus) captured in six counties in Illinois between May 2002 and February 2004 for West Nile virus (WNV) antibodies. One female big brown bat tested positive for WNV antibodies. Samples of kidney, liver, and heart tissue were collected from 312 bats of seven species that were submitted to the Illinois (USA) Department of Public Health or the Illinois Department of Agriculture diagnostic laboratories between January 2001 and December 2003. Tissue samples were tested for WNV using TaqMan reverse transcriptase polymerase chain reaction and all were negative. Prevalence of WNV antibodies in the bats (1%) was lower than previously reported for other flaviviruses, but similar to the prevalence (2%) of WNV antibodies reported in bats from New Jersey and New York, USA. Additional research is needed to determine potential impact of WNV infections on bats and to determine whether they play a role in the WNV transmission cycle.