During T cell development in the thymus, a certain population of self-reactive thymocytes differentiates into regulatory T cells that suppress otherwise harmful self-reactive T cells. In transgenic mice expressing both TCR that specifically recognizes moth cytochrome c and the moth cytochrome c ligand, a large proportion of CD4+ T cells expresses CD25 and secretes TGF-β1 upon Ag stimulation. Because TGF-β1 expression by these T cells can be decreased by cyclosporin A, a NF-AT inhibitor, NF-AT-mediated TGF-β1 expression in T cells was addressed by characterizing a NF-AT response element in the TGF-β1 promoter. Analysis of the mouse TGF-β1 promoter (-1799 to +793) in transfection experiments in T cell 68-41 hybridoma cells detected NF-AT binding sites at positions +268 and +288 in the proximal promoter region. Binding of NF-AT to this region was detected only in tolerant CD4+ T cells, but not in fully activated CD4+ T cells by chromatin immunoprecipitation assays. Activation of these NF-AT sites was sufficient to induce TGF-β1 promoter activity; however, additional signaling due to full Ag stimulation blocked NF-AT-mediated TGF-β1 expression. This suppression of the TGF-β1 promoter is mediated by the -1079 to -406 region, in which deletion of a GATA-binding motif at position -821 abrogates NF-AT-mediated activation of the TGF-β1 promoter. Therefore, TGF-β1 expression in T cells is controlled by multiple regulatory factors that have distinct functions in response to partial or full TCR activation.