Sodium dodecyl sulphale polyacrylamide gel elcctrophoresis (SDS-PAGE) and quantitative immunoelectrophoretic techniques have been used to characterize further the two major mouse allergens, antigen I (Ag 1) and antigen 3 (Ag 3). Gel filtration using Sephacryl S-200 showed Ag 1 to have a molecular weight of 18 kD and Ag 3 of 21 kD. SDS-PAGE followed by Western blotting onto nitrocellulose then incubation with individual antisera directed against each of the two major allergens, and an alkaline phosphalase enzyme system, was used to distinguish between the two allergens and indicated a molecular weight of 17 kD for Ag 1 and 16 kD for Ag 3. Ag 3 but not Ag 1 was shown to contain polysaccharide residues. Immunohistochemical staining of mouse skin sections demonstrated that antigens detected in whole dust extracts were present in the hair follicles, on the hair shafts and on the stratum corneum. Staining of similar sections using the rabbit anti-Ag 3 showed the presence of this major allergen in the hair follicles coaling the hairs and extending along the skin surface. Serum from a pool of mouse-allergic subjects also demonstrated staining in the same areas when detected using a fluoroscein-labelled anti-human IgE as second antibody. As both major allergens were present in extracts of fur this would appear to be most appropriate for use in diagnosis (i.e. skin test and RAST) and also possibly desensitization. However, dust from isolators (available in greater amounts) would be equally suitable.