On the basis of expression of the T cell differentiation antigen CD27, human peripheral blood CD4⁺ memory cells can be divided into two subsets, a large CD45RA⁻CD27⁺ (82%) and a small CD45RA⁻CD27⁻ (18%) population. Analysis of the functional properties of these memory T cell subsets showed that proliferative responses to the recall antigen tetanus toxoid (TT), shortly after booster immunization, were mainly confined to the CD27⁻ population. Also, in atopic individuals, proliferative responses to allergens for which these individuals are sensitized, were limited to the CD45RA⁻CD27⁻ population. After stimulation with CD3 monoclonal antibody and phorbol ester, CD27⁺ cells produced vast amounts of interleukin (IL)‐2 but minimal amounts of IL‐4, whereas in marked contrast, CD27⁻ T cells secreted low levels of IL‐2 and high levels of IL‐4. The capacity of the vast majority of these latter cells to produce IL‐4 was found to be a stable feature since high IL‐4‐secreting T cell clones were generated from the CD27⁻ subset. These findings suggest that upon renewed as well as chronic antigenic stimulation in vivo, memory T cells acquire the CD45RA⁻CD27⁻ phenotype and that, as a consequence, in this subset functionally differentiated CD4⁺ T cells are compartmentalized. Our results predict that analysis of the small CD27⁻ subset of memory cells, that makes up approximately 10% of the peripheral blood T cell population, will provide information on the specificity and function of responding CD4⁺ T cells at a given point in time in healthy and diseased individuals.