Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)(2)D(3) is a secosteroid hormone that activates the nuclear receptor vitamin D(3) receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-gamma and IL-12 genes by 1,25(OH)(2)D(3) in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)(2)D(3) on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)(2)D(3) inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)(2)D(3)-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)(2)D(3), and confirm the interest for a possible pharmacological use of this molecule and its derivatives.