A double monoclonal antibody-based assay for the 70-kD Alternaria allergen GP70 is described. The assay is sensitive to GP70 concentrations as low as 0.2 microgram/mL and is highly specific for this allergen. The glycoprotein detected by this assay is shown to bind to human IgE from Alternaria-sensitive patients, proving that it is an allergen. Two of three commercial Alternaria preparations tested contained no detectable GP70. Several related fungi were shown to contain detectable concentrations of GP70, but the most abundant source was a commercial preparation of house dust. Further measurement of GP70 in biologic materials should increase our knowledge of the distribution and importance of this Alternaria allergen in the environment and in the development of allergic diseases.