Background: Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results. Objective: We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures and to examine the role of protease inhibitors in enhancing allergen stability. Methods: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at –70°C, –20°C, 4°C, or 37°C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immunoblot analyses. Results: The overall potencies of the stored extracts measured by competition ELISA were stable at –20°C and 4°C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4°C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37°C led to overall loss of potency and allergen content, whereas storage at –70°C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability. Conclusion: Although overall potency of the extracts, as measured by competition ELISA, was preserved at –20°C and 4°C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens. (J Allergy Clin Immunol 2000;105:482-8.)