IntroductionThe current view is that interferon γ (IFNγ) activates cells via interaction with the extracellular domain of the receptor complex (reviewed in Stark et al., 1998). This in turn results in the activation of the receptor-associated tyrosine kinases JAK1 and JAK2, leading to phosphorylation and dimerization of the transcription factor STAT1α, which dissociates from the receptor cytoplasmic domain and undergoes nuclear translocation. This current view ascribes no further role to IFNγ or the receptor chains IFNGR1 and IFNGR2 in IFNγ signaling. The above scenario ignores several events associated with IFNγ signaling. First, both IFNγ and one of the receptor chains, IFNGR1, undergo internalization or endocytosis and nuclear translocation Subramaniam et al., 2000). Second, at least three separate studies showed that intracellular IFNγ possessed biological activity that was qualitatively not unlike that of extracellular IFNγ. In one, microinjection of human IFNγ into mouse macrophage cells induced Ia expression (Smith et al., 1990). Extracellularly added human IFNγ did not have an effect, since it does not recognize the extracellular domain of the mouse IFNγ receptor complex. Another study involved internalizing human IFNγ into mouse macrophages via a liposomal delivery system, resulting in induction of an antiproliferative effect (Killion et al., 1994).Finally, intracellular expression of the human IFNγ gene in mouse fibroblasts in a non-secretory form resulted in induction of antiviral activity (Sanceau et al., 1987).Recently, we have determined the structural basis for nuclear translocation of murine IFNγ by identification of a polycationic nuclear localization sequence in its C-terminus (Subramaniam et al., 1999). Further, internalized IFNγ binds to the cytoplasmic domain of the IFNGR1 receptor chain . Immunoprecipitation experiments showed that a cytoplasmic complex of IFNγ-IFNGR1-STAT1α complexed to the nuclear importin α protein, NPI-1, occurs in an NLSdependent fashion . Thus, the internalization of IFNγ and nuclear transport of IFNγ and IFNGR1 appear to be a mechanism for nuclear import of the IFNγ transcription factor STAT1α.In the present study, we have expressed a non-secretable form of human IFNγ and found it to be biologically active. In order to demonstrate that the NLS of IFNγ was key to the intracellular events described above, positively charged amino acids in the NLS were replaced with alanines, such that the NLS sequence 128 KTGKRKR 134 was mutated to 128 ATGAAAA 134 . Non-secreted forms of IFNγ or its NLSmutated version were tested in murine and human cells for their ability to induce IFNγ activity, to activate STAT1α and to carry out nuclear translocation of STAT1α. The data show that