Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to upregulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the β chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-κB, β E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription. The failure to induce NF-AT completely may be a consequence of a diminished calcium flux, since the PKC pathway was apparently intact. It was found that the calcium ionophore ionomycin could either induce anergy or abrogate the induction of nonresponsiveness according to the dose, also suggestive of differences in calcium signaling. The pattern of expression of transcription factors during the induction of T-cell anergy is consistent with the inability of anergic cells to produce IL-2. These results demonstrate that there are differences in the early nuclear events characteristic of stimuli, the outcome of which leads to cells that are phenotypically similar, but are functionally different.