HLA-G displays immunotolerogenic properties towards the main effector cells involved in graft rejection through inhibition of NK- and CTL-mediated cytolysis and CD4+ T cell alloproliferation. HLA-G expression is restricted in healthy tissues to trophoblast and thymus but is extended to various tissues under pathological conditions. HLA-G was detected in allograft biopsies and sera from transplanted patients who displayed a better graft acceptance. However, the cells involved in such de novo expression of HLA-G remain to be characterized. By flow cytometry and confocal microscopy, we demonstrated that, following allogeneic stimulation in vitro, both CD4+ and CD8+ T cell subsets can express membrane-bound HLA-G1 and/or soluble HLA-G5 molecules. Such HLA-G1/-G5 expression is regulated at the transcriptional level. Soluble HLA-G5 could be detected by using a novel monoclonal antibody, 5A6G7, specific for the intron 4-retaining sequence of HLA-G5. Finally, the biological relevance of these data was provided by analysis of transplanted patients in whom we identified both CD4+ and CD8+ T cells expressing HLA-G. The HLA-G-positive T cells we describe here may constitute a cellular source of HLA-G after allotransplantation and may be involved in the improved graft acceptance which is observed in HLA-G-positive transplanted patients.