The phenotype and activation status of leukocytes infiltrating human late phase allergic skin reactions were investigated by immunocytochemistry using a panel of mAb. Late phase skin reactions were induced in atopic individuals by intradermal challenge with allergen extract (grass pollen or house dust mite) and skin biopsies were obtained 6, 24, or 48 h after challenge. Cryostat sections were examined for evidence of infiltration and activation of T cells and eosinophils. Biopsies from saline-challenged control sites were used for comparison. A substantial number of CD3+ cells were observed close to the dermal capillaries. The number of CD4+ cells showed a similar pattern but few CD8+ cells were seen. The ratio of CD4 and CD8 subsets in tissue did not relate to the CD4/CD8 ratio in the peripheral blood. Two observations suggested that T cells had become activated: first, a small number of infiltrating cells bore receptors for IL-2, and second, there was indirect evidence of IFN-gamma secretion, demonstrated by increased expression of HLA-DR on endothelial cells and de novo expression of CD4 Ag on epidermal Langerhans cells. Activated eosinophils were detected in most of the allergen-challenged biopsies. The late phase reaction diameter at 6 h correlated with the number of activated eosinophils at 48 h (r = 0.61, p = 0.05), but not with infiltration by T cells. There was a strong correlation between the numbers of CD4+ cells and activated eosinophils at 24 h (r = 0.94, p less than 0.001). These findings suggest that interactions between T lymphocytes and eosinophils in reactions to airborne allergens may be more important than has previously been recognized.