Although mite major group I allergens, Der p I and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p I exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p I or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group I allergens and mite-derived serine proteases. Recombinant mite group I allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p I and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group I allergens. (C) 2005 Elsevier Inc. All rights reserved.