The allergenicity and enzyme content of extracts of Dermatophagoides pteronyssinus whole mite, cuticle (CUT), and spent-growth medium (SGM) used in its culture have been examined. Both direct RAST binding and RAST-inhibition studies demonstrated that the whole mite extract was approximately threefold more potent than either the CUT or SGM extracts. Crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis demonstrated that whole mite extracts contained 36 antigens of which nine were judged to be allergens (Dpt 1, 3, 4, 7, 8, 12, 17, 22, and 31). In contrast, extracts of CUT and SGM both contained 15 mite antigens of which five were judged to be allergens (Dpt 4, 8, 12, 17, and 22). Enzyme studies demonstrated that whole mite extracts from various sources contained esterases, phosphatases, aminopeptidases, and glycosidases. Both the CUT and SGM extracts contained a similar spectrum of enzymes, although the glycosidases were present at relatively low levels. Combined crossed immunoelectrophoresis and enzymic studies identified three antigens (Dpt 6, 13, and 24) with esterase activity, one antigen (Dpt 27) with beta-glucuronidase activity, and one antigen (Dpt 14) with acid phosphatase activity. Thus far, none of these enzymes has been demonstrated to be allergenic. Both enzymic and immunochemical studies suggested that not all mite allergens are of an excretory/secretory origin, but some (e.g., Dpt 1, 3, 4, 7, and 31) are derived from the whole mite per se.