CD27, a lymphocyte-specific member of the TNF/NGF-R family, is expressed on the majority of peripheral blood T cells. Activation of T cells via TCR/CD3 induces high CD27 surface expression and the release of a soluble extracellular part of the molecule. After prolonged activation in vitro, CD27 becomes gradually switched off. There is evidence that also in vivo, CD4+ cells that have persistently been stimulated by Ag, accumulate within the CD45RA-CD27- subset. In addition, an increase of CD27- T cells has been observed under certain immunopathologic conditions and during aging. This study was undertaken to analyze the regulation of CD27 on different T-cell subsets and to determine whether the loss of CD27 expression is an irreversible event and may thereby mark T-cell differentiation. In agreement with earlier findings, all CD4+CD45RA+CD45RO- T cells were found to express CD27, whereas a small fraction of the CD4+CD45RA-CD45RO+ subset lacks the molecule. In contrast, within the CD8+ compartment CD27- subsets were found both in the CD45RA+ and CD45RA- subpopulations. After stimulation with CD3 mAb, both CD27 membrane expression and release was equally up-regulated in CD4+ and CD8+ subpopulations. This stimulus, however, provoked a strikingly predominant up-regulation of membrane CD27 on CD45RO- cells as compared with CD45RA- cells. On CD4+CD45RA-CD27- T cells and long-term grown CD45RA-CD27- TLC, CD27 expression could not be reinduced after stimulation of the TCR/CD3 complex, neither at the protein nor at the mRNA level. Comparison of CD27 expression with its structural homologue FAS/APO-1 showed that down-regulation after prolonged activation is not a general feature of TNF/NGF-R family members. The CD27 ligand was recently identified and was shown to give a co-stimulatory signal to PHA-activated T cells. The restricted up-regulation of CD27 on CD45RA+ cells after T-cell stimulation may point at a discrete role of CD27-CD27 ligand interaction during transition of CD45RO- to CD45RA- T cells. In addition, the CD27 negative phenotype seems a stable reflection of differentiation rather than of activation.