West Nile fever caused fatal disease in humans, horses, and birds in the northeastern United States during 1999. We studied birds from two wildlife facilities in New York City, New York, that died or were euthanatized and were suspected to have West Nile virus infections. Using standard histologic and ultrastructural methods, virus isolation, immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction, we identified West Nile virus as the cause of clinical disease, severe pathologic changes, and death in 27 birds representing eight orders and 14 species. Virus was detected in 23/26 brains (88%), 24/ 25 hearts (96%), 15/18 spleens (83%), 14/20 livers (70%), 20/20 kidneys (100%), 10/13 adrenals (77%), 13/ 14 intestines (93%), 10/12 pancreata (83%), 5/12 lungs (42%), and 4/8 ovaries (50%) by one or more methods. Cellular targets included neurons and glial cells in the brain, spinal cord, and peripheral ganglia; myocardial fibers; macrophages and blood monocytes; renal tubular epithelium; adrenal cortical cells; pancreatic acinar cells and islet cells; intestinal crypt epithelium; oocytes; and fibroblasts and smooth muscle cells. Purkinje cells were especially targeted, except in crows and magpies. Gross hemorrhage of the brain, splenomegaly, meningoencephalitis, and myocarditis were the most prominent lesions. Immunohistochemistry was an efficient and reliable method for identifying infected cases, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses. In contrast, the in situ hybridization probe pWNV-E (WN-USAMRIID99) reacted only with West Nile virus. These methods should aid diagnosticians faced with the emergence of West Nile virus in the United States.