Interferons (IFNs) are a family of hormone-like secretory proteins with multiple phenotypical changes, including gene expression and morphological alterations. Earlier studies have shown that IFN-activated Tyk2 kinase physical associates with p95Vav (Vav), a proto-oncogene gene product expressed in hematopoietic cells. Since Tyk2 is a cytoplasmic kinase and Var is believed to be localized in the nuclear compartment, here we explored the possibility of Vav redistribution in IFN-alpha-activated cells, using the U266 human myeloma cell Line as a model system. Using biochemical assays and in situ confocal microscopy; we demonstrate that IFN-alpha treatment triggers a rapid (10 min) translocation of Vav from the nuclear compartment to the cytoplasm. In addition, we also show the existence of IFN-alpha-induced physical interaction between Vav and Ku80, Ku80, and Tyk2, and among Vav Ku80, and Tyk2 in the cytoplasmic compartment of IFN-stimulated cells. The observed IFN-alpha-induced association among Vav, Ku80, and Tyk2 was dependent on cellular tyrosine kinase activity. Since recently Vav has been shown to promote the GDP/GTP exchange activity of the cytoskeleton signaling molecule small GTPase Rad and activates its downstream signaling, our present findings raise the possibility of involvement of the small GTPase in IFN signaling leading to its biological effects, including cytoskeleton reorganization.