In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin ␣ in the presence of GTPbound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand b) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C-adjacent region (b-c loop region) of the strand b became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.The STAT (signal transducers and activators of transcription) family proteins (STAT1 to -4, -5A, -5B, and -6) are phosphorylated by cytokine stimulation, form homo-or heterodimers, and enter the nucleus, where they regulate expression of their target genes (6, 13). STATs have a variety of functions, including antiapoptosis, proliferation, differentiation, inflammation, and development under physiological conditions, and of note, the oncogenic activities of STAT3 and STAT5 have also been demonstrated under pathological conditions (5).A small GTPase Rac1 is implicated in cytoskeletal organization, membrane ruffling, production of superoxide, phagocytosis, and chemotaxis as well as regulation of the cell cycle (15, 39). Recent studies revealed its distinct roles in nuclear translocation of phosphorylated STATs (p-STATs) and -catenin and also its nuclear accumulation in the G 2 phase, promoting cell division (17,31,47). MgcRacGAP is an evolutionarily conserved GTPase-activating protein (GAP) for Rho family GTPases. We and others previously showed that MgcRacGAP controls the mitotic spindle through associating ␣-, -, and ␥-tubulin, Rho family GTPases, and a kinesin protein, MKLP1, and plays essential roles in the completion of cytokinesis, accumulating to the midbody (12, 16, 32). Very recently, Yamada et al. reported that conditional knockout of MgcRac-GAP results in acute apoptosis even before the failure of cytokinesis in interleukin-7 (IL-7)-expanded B220 ϩ cells (48), indicating that MgcRacGAP is not simply involved in cell division but also in cell survival, at least in IL-7-expanded B220 ϩ cells.Molecular trafficking between the nucleus and cytoplasm occurs via nuclear pore complexes. To enter the nucleus, nuclear proteins larger than ϳ50 kDa usually harbor a functional nuclear localization signal (NLS) or bind NLS-co...