Recent investigations support the idea that angiogenesis is involved in the pathophysiology of leukemia. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein showing angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. The aim of the present study was to establish a recombinant adenovirus (ADV.TSP-1(f)) expressing antiangiogenic fragment of TSP-1 (TSP-1(f)), and to determine the feasibility for use of the adenovirally expressed TSP-1(f) in leukemia gene therapy. The results of this investigation showed that TSP-1(f) was expressed efficiently in adenovirus-transduced human myelogenous leukemia K562 cells. Compared to the controls, although there was almost no effect on proliferation of K562 cells in vitro, adenovirus-mediated TSP-1(f) transduction inhibited the growth of K562 xenografts dramatically. Furthermore, the microvessel density (MVD) was much lower in the ADV.TSP-1(f)-treated tumors compared to the controls. These data support the use of in vivo gene delivery approach to produce antiangiogenic fragment of TSP-1 for leukemia therapy.