The NF-AT family of transcription factors participates in the regulation of early immune response genes such as IL-2, IL-4, CD40 ligand, and Fas ligand in response to Ca2+/calcineurin signals initiated at the antigen receptor. Calcineurin activation leads to the rapid translocation of NF-AT family members from cytoplasm to nucleus, an event that is blocked by the immunosuppressive drugs cyclosporin A and FK506. We show that translocation requires two redundant nuclear localization sequences and that one sequence is in an intramolecular association with phosphorserines in a conserved motif located at the amino terminus of each NF-AT protein. Mutation of serines in this motif in NF-ATc both disrupts this intramolecular interaction and leads to nuclear localization, suggesting a model of NF-AT nuclear import in which dephosphorylation by calcineurin causes exposure of two nuclear localization sequences.