Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.