Effects of adenoviruses carrying genes of a vascular endothelial growth factor (vegf 165), a glial-cell derived neurotrophic factor (gdnf), and angiogenin (ang) were studied in a rat model of chronic hindlimb ischemia. Therapeutic genes were used for direct gene therapy in the following combinations: (1) Ad5-ANG; (2) Ad5-VEGF+Ad5-ANG; and (3) Ad5-VEGF+Ad5-ANG+Ad5- GDNF. Real-time PCR demonstrated increased gdnf, vegf, and ang mRNA levels within the area of an ischemic muscle on day 14 where the study combinations of therapeutic genes were injected in both Ad5-VEGF+Ad5-ANG and Ad5-VEGF+Ad5- ANG+Ad5-GDNF groups. On post-injection day 28, the number of centronuclear myotubes (CNMs) as well as the CD31- immunopositive cell count showed a 38.7- and 1.3-fold increase, respectively, within the ischemic area in the Ad5-VEGF+Ad5- ANG+Ad5-GDNF group compared with the Ad5-VEGF+Ad5-ANG group. There was an increased expression of desmin mRNA in the area of an ischemic muscle in Ad5-VEGF+Ad5-ANG and Ad5-VEGF+Ad5-ANG+Ad5-GDNF groups on day 14. Based on Western blotting results, the expression of CD34 and a von Willebrand factor (VWF) increased on day 14 after injection of Ad5-VEGF+Ad5-ANG+Ad5-GDNF as compared with the control group. The Ad5-VEGF+Ad5-ANG+Ad5-GDNF injection stimulates muscle regeneration by increasing the number of CNMs and blood vessels. Based on the above findings, the gdnf angiogenic and regenerative potential necessitates further studies of its possible use as an agent stimulating angiogenesis and skeletal muscle regeneration for clinical purposes.