A significant activity to elicit histamine release was found in an ultrafilterable (Mr-cutoff < 10 kD) fraction of a mite feces extract from a spent mite medium. The activity was divided into two fractions (LM1 and LM2) on an Ultrogel AcA 54 column when monitored by histamine release assay using washed peripheral blood cells from mite-allergic patients. The larger-molecular-size antigen, LM1 was further separated into three allergenic fractions by consecutive chromatography on Sephadex G-50, GM-DEAE, and TSKgel ODS-120T. All the purified allergens, LM1s (LM1a, LM1b, and LM1c), which produced positive reactions in skin tests on allergic patients, were glycoproteins (molecular weight: 8 kD on SDS-PAGE; 12.5 kD on Gel filtration) with different carbohydrate contents and pI values ranging from 4 to 5 on an IEF plate. LM1s were cross-reactive with anti-Der f II but not with anti-Der f I. The reaction of LM1c to anti-Der f II serum was completely inhibited with the other antigens, while the reactions of LM1a and LM1b to the serum were partially inhibited with LM1c or the other antigens, respectively.