Understanding the uses and limitations of methods for rapid diagnosis of fungal disease is essential in order to diagnose and treat these infections early in their course. Antigen detection methods are useful for diagnosis of aspergillosis, cryptococcosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, and penicilliosis marneffei. The accuracy of the beta-glucan assay for diagnosis of aspergillosis and candidiasis and its role in fungal diagnosis remains unclear, in part because the few published studies report widely varying specificity. Serologic tests for antibodies are also useful for diagnosis of histoplasmosis and coccidioidomycosis, but their sensitivity may be reduced by immunosuppression. While molecular diagnostic methods have been described and are available at some reference and university laboratories, their role in patient care remains uncertain, largely because of the lack of well-characterized assays and studies establishing their accuracy. Culture methods, although essential for establishing the diagnosis in some cases, have limitations for rapid diagnosis, namely insensitivity, need for invasive procedures, and delayed growth.