NFATc (a member of the family of nuclear factors of activated T cells) is a transcriptional factor responsible for the Ca(2+)-inducible activation of cytokine genes during the immune response. In resting T cells, NFATc is retained in the cytoplasm by a mechanism that depends on multiple phosphorylations in an N-terminal regulatory domain. Physical interaction with and dephosphorylation by Ca(2+)-activated calcineurin (Cn) allows the protein to enter the nucleus, where it binds to specific sites in cytokine gene promoters. Previous studies had identified a peptide segment in NFATc that binds Cn stably. Here we report the identification of a second Cn-binding element in NFATc, which synergizes with the previously identified element. Although these sequences are conserved in all isoforms of NFAT, we find that the two sites contribute differentially to the overall affinity for Cn in an isoform-dependent manner. The regulatory domain of NFAT also was found to be entirely devoid of structure, both in the phosphorylated and unphosphorylated state. This finding suggests that the NFAT regulatory domain does not undergo phosphorylation-induced conformational switching, but instead requires partner proteins to control accessibility of the NFAT nuclear localization and nuclear export signals.