An outbreak of West Nile virus (WNV) in and around New York City during the late summer of 1999 was the cause of extensive mortality among free-ranging birds. Within the Bronx Zoo/Wildlife Conservation Park, viral activity was also observed and produced some morbidity and mortality among specimens in the zoo's bird collection and probably caused morbidity in at least one specimen from the zoo's mammal collection. To determine the extent of the outbreak and attempt to ascertain the temporal appearance of virus within the park, a serologic survey of birds and mammals was performed. The survey showed that 34% of tested birds (125 of 368; 124 species) were positive for antibody to WNV. The virus caused a disease to infection ratio of 22% (27 of 125) among birds with a 70% (19 of 27) case fatality rate. In contrast, only 8% of the mammals (9 of 117; 35 species) possessed antibody to WNV and there was no virus-associated mortality. Testing of banked and fresh sera obtained from both birds and mammals revealed that there was no evidence of WNV circulation before the 1999 outbreak and that birds introduced into the park were not the source of the New York outbreak. West Nile virus RNA was detected in tissues from one bird that died in February 2000, long after the end of the mosquito transmission season. The potential importance of zoologic parks as possible sentinels for emerging diseases is discussed.