In the cell nucleus the NS1 protein of influenza A virus inhibits both pre-mRNA splicing and the nuclear export of mRNAs. Both the RNA-binding and effector domains of the protein are required for these nuclear functions. Here we demonstrate that the NS1 protein has a latent nuclear export signal (NES) that is located at the amino end of the effector domain. In uninfected, transfected cells the NS1 protein is localized in the nucleus because the NES is specifically inhibited by the adjacent amino acid sequence in the effector domain. Substitution of alanine residues for specific amino acids in the adjacent sequence abrogates its inhibitory activity, thereby unmasking the NES and causing the full-length NS1 protein to be localized to the cytoplasm. In contrast to uninfected cells, a substantial amount of the NS1 protein in influenza virus-infected cells is located in the cytoplasm. Consequently, the NES of these NS1 protein molecules is unmasked in infected cells, indicating that the NS1 protein most likely carries out functions in the cytoplasm as well as the nucleus. A dramatically different localization of the NS1 protein occurs in cells that are infected by a virus encoding an NS1 protein lacking the NES: the shortened NS1 protein molecules are almost totally in the nucleus. Because the NES of the full-length NS1 protein is unmasked in infected but not uninfected cells, it is likely that this unmasking results from a specific interaction of another virus-specific protein with the NS1 protein.