A new set of allergens from Dermatophagoides pteronyssinus and D. farinae (provisionally named DPS and DF5, respectively) was isolated from the whole culture of mites. The apparent molecular weights of both allergens were shown to be 25000 on SDS-PAGE under a reducing condition and 27000 on Sephadex G-75 gel nitration chromatography. Both DP5 and DF5, as well as Der f III, possessed proteolytic activity. The results of substrate specificity and susceptibility to various protease inhibitors of DP5 and DF5 strongly suggested that they belonged to the chymotrypsin-like serine protease family. In sera from 88 mite-allergic patients, specific IgE antibodies to DP5 and/or DF5 were detected in only 41% of the sera by radio-allergosorbent test, while 90% and 93% had specific IgE antibodies to Der p I and/or Der f I and Der p II and/or Der f II, respecti vely.