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We evaluated phenotype and apoptotic status of normal CD4+CD69+ and CD8+CD69+ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4+ and CD8+ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25negCD38negTCRalphabetapos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69+ T-cells. A considerable proportion of CD69+ lymphocytes expressed intracellular perforin; in addition, an average 16+/-6% CD69+ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69+ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38negCD25negTCRalphabetapos T-lymphocytes.