West Nile virus (WNV), a mosquito-borne flavivirus introduced recently to North America, is a human, equine, and avian neuropathogen. The majority of human infections with WNV are mosquito-borne; however, laboratory-acquired infections with WNV and other arboviruses also occur. This report summarizes two recent cases of WNV infection in laboratory workers without other known risk factors who acquired infection through percutaneous inoculation. Laboratory workers handling fluids or tissues known or suspected to be WNV-infected should minimize their risk for exposure and should report injuries and illnesses of suspected occupational origin to their supervisor.