Hairy cell leukemia is an uncommon B-cell lymphoproliferative disease of unknown etiology in which tumor cells display characteristic microfilamentous membrane projections. Another striking feature of the disease is its exquisite sensitivity to interferon (IFN)-alpha. So far, none of the known IFN-alpha regulatory properties have explained IFN-alpha responsiveness nor have they taken into account the morphological characteristics of hairy cells. IFN-alpha profoundly alters cytoskeletal organization of hairy cells and causes reversion of the hairy appearance into a rounded morphology. Because cytoskeletal rearrangements are controlled by the Rho family of GTPases, we investigated the GTPase activation status in hairy cells and their regulation by IFN-alpha. Using immunolocalization techniques and biochemical assays, we demonstrate that hairy cells display high levels of active Cdc42 and Rac1 and that IFN-alpha down-regulates these activities. In sharp contrast, RhoA activity was low in hairy cells but was increased by IFN-alpha treatment. Finally, IFN-alpha-mediated morphological changes also implicated a p53-induced response. These observations shed light on the mechanism of action of IFN-alpha in hairy cell leukemia and are of potential relevance for the therapeutical applications of this cytokine.