Monoclonal antibody affinity chromatography was used to purify two homologous mite allergens, Der f II from Dermatophagoides farinae and Der p II from D. pteronyssinus. They have the same molecular weight (MW) (15 kd) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they have similar amino acid compositions, and their N-terminal amino acid sequences differ in only four of the first 35 residues. An excellent correlation was observed between IgE antibody to Der f II and Der p II measured in sera from 65 mite-allergic patients (r = 0.94; p less than 0.001) and between quantitative intradermal skin tests to both allergens. A third allergen (Der f III, MW 29 kd) was purified from D. farinae by repeated gel filtration. In sera from 51 mite-allergic patients, IgE antibody to Der f II, Der f III, and previously purified Der f I (MW 24 kd) was detected in 92%, 16%, and 78% of the sera by radioimmunoassay, respectively. Most patients, 41/51 (80%), demonstrated IgE antibody to more than one allergen. With monoclonal antibodies fully cross-reactive with Der f II and Der p II, a two-site immunoassay was developed for measuring absolute quantities (nanograms or micrograms) of these allergens. In extracts rich in mite-fecal material (n = 5), Der f I and Der p I (group I allergens) and Der f II and Der p II (group II allergens) were measured in ratios of 11:1 to 35:1. Lower ratios (1.1:1 to 7:1) were observed in mite body extracts (n = 6). These experiments clearly define a second group of major dust mite allergens that demonstrate extensive structural and antigenic homology.