The mechanism underlying positive patch tests with house dust mite-allergen, Dermatophagoides pteronyssinus (Der p), in patients with atopic dermatitis was investigated by isolating T cells from the test sites of two patients. Eighty-five T cell clones (TCC) were established from the epidermis and dermis of lesional skin by the limiting-dilution method with Der p and interleukin (IL)-2. With restimulation assays, 29 of 60 TCCs tested demonstrated specific proliferation; 85% were of the CD3+, CD2+, and CD4+ phenotype. Der p-specific T cells constituted 0.4% to 2.7% of lesional T cells, and they were more frequent in the skin than in the blood of the patients by one order of magnitude. The mitogen-stimulated lymphokine profile of 55 TCCs was assessed; 42% (11/26) of the allergen-specific TCCs secreted IL-4 but almost no interferon-gamma, as described for the Th2 subset of the mouse. Also, six selected TCCs supported IgE secretion by autologous lymphocytes. Only three of 26 allergen-specific, skin-derived TCCs demonstrated a Th1-like lymphokine profile. These results support the specific nature of Der p-induced patch test lesions in patients with atopic dermatitis, and the results demonstrate also that a considerable proportion of lesional T cells are allergen-specific, IL-4-producing T cells that are capable of enhancing IgE production.