Stat3 is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation is unclear. In this study, by systematic deletion and site-directed mutagenesis we identified Arg-214/215 in the ␣-helix 2 region of the coiled-coil domain of Stat3 as a novel sequence element essential for its nuclear translocation, stimulated by epidermal growth factor as well as by interleukin-6. Furthermore, we identified Arg-414/417 in the DNA binding domain as also required for the nuclear localization of Stat3. This sequence element corresponds to Lys-410/413 of Stat1, a reported sequence for Stat1 nuclear translocation. On the other hand, Leu-411 of Stat3, corresponding to Leu-407 of Stat1, a necessary residue for Stat1 nuclear transport, is not essential for Stat3 nuclear import. The mutant of Arg-214/215 or Arg-414/417 was shown to be tyrosyl-phosphorylated normally but failed to enter the nucleus in response to epidermal growth factor or interleukin-6. The defect, however, can be rescued by the wild-type Stat3 but cannot be compensated by these two mutants. Mutations on Arg-414/417, but not Arg-214/215, destroy the DNA binding activity of Stat3. Our data for the first time identified a sequence element located in the coiled-coil domain that is involved in the ligand-induced nuclear translocation of Stat3. This novel sequence together with a conserved sequence element in the DNA binding domain coordinates to mediate the nuclear translocation of Stat3.In eukaryotic cells, certain latent transcription factors are primarily located in the cytoplasm under unstimulated conditions. Upon an extracellular stimulation, however, they are rapidly translocated into the nucleus, where they bind to the regulatory elements of the target genes and regulate their expression. Some of these factors are subsequently exported to the cytoplasm. The nuclear import and export of the transcription factors therefore function as an important biological switch for the control of gene expression and cellular responses. In general, proteins to be imported contain a specific target sequence designated NLS, 1 a nuclear localization signal, consisting of either a stretch of basic amino acids or two basic stretches separated by a spacer ranging from 10 to 37 amino acids (1, 2). These proteins bind to a cytoplasmic receptor composed of importin ␣ and importin . Importin ␣ belongs to a divergent family of proteins that serves as adaptors between the substrates and importin  (3, 4). Importin  docks the NLS-containing protein and the importin ␣ complex at the nuclear pore complex, which is a specialized structure of the nuclear envelope (5-8). The complex is translocated into the nucleus through the nuclear pore complex in an energy-dependent manner (9).STATs are a family of latent cytoplasmic transcription factors that were named by virtue of their novel and unique dual functions as signaling molecules in the cytoplasm and as transcription factors...