A multiyear study was conducted using an enzyme-linked immunosorbent assay to measure antibody to address the application of the test to the diagnosis of aspergillosis in avian species. In general serostudies (n  =  1314), four avian groups (psittaciform, raptor, penguin, and zoo) were found to have samples with antibody reactivity. Penguin, raptor, and zoo groups were found to have higher levels of antibody to Aspergillus than the psittaciform group. Additional clinical information was collected on 303 cases, which resulted in the definition of presumptive normal, probable, and confirmed infection groups. Although the confirmed group was more likely to have antibody reactivity, the mean antibody index was not found to be significant between presumptive normal and probable or confirmed cases.