Definitive diagnosis of invasive aspergillosis often requires tissue samples for histologic evidence of fungal infection and culture confirmation of Aspergillus species. However, the culture frequently fails to isolate Aspergillus species. Alternative approaches to confirm Aspergillus infection use polymerase chain reaction, in situ hybridization, and immunohistochemical analysis on paraffin-embedded sections. These approaches are well characterized in animals and adult patients but not pediatric patients. We studied the immunoreactivity of a commercially available monoclonal antibody, Mab-WF-AF-1 (DAKO, Carpinteria, CA), on paraffin-embedded sections from 16 pediatric cases with invasive aspergillosis, of which 12 were proven by culture. Optimal immunoreactivity required microwave antigen retrieval using high pH; 5 other antigen retrieval approaches were unsuccessful. With optimization, the monoclonal antibody was strongly immunoreactive in all cases with staining of the Aspergillus cell wall, septa, and cytoplasm. Background was minimal with no cross-reactivity to Candida albicans. These findings demonstrate the usefulness of the Mab-WF-AF-1 antibody in pediatric tissues suspected of invasive aspergillosis.