We reported the therapeutic efficacy of (131)I in hepatocellular carcinoma (HCC) cells stably expressing the sodium iodide symporter (NIS) under the control of the tumor-specific α-fetoprotein (AFP) promoter. In the current study we investigated the efficacy of adenovirus-mediated in vivo NIS gene transfer followed by (131)I and (188)Re administration for the treatment of HCC xenografts. We used a replication-deficient adenovirus carrying the human NIS gene linked to the mouse AFP promoter (Ad5-AFP-NIS) for in vitro and in vivo NIS gene transfer. Functional NIS expression was confirmed by in vivo γ-camera imaging, followed by analysis of NIS protein and mRNA expression. Human HCC (HepG2) cells infected with Ad5-AFP-NIS concentrated 50% of the applied activity of (125)I, which was sufficiently high for a therapeutic effect in an in vitro clonogenic assay. Four days after intratumoral injection of Ad5-AFP-NIS (3×10(9) plaque-forming units) HepG2 xenografts accumulated 14.5% injected dose (ID)/g (123)I with an effective half-life of 13 hr (tumor-absorbed dose, 318 mGy/MBq (131)I). In comparison, 9.2% ID/g (188)Re was accumulated in tumors with an effective half-life of 12.8 hr (tumor-absorbed dose, 545 mGy/MBq). After adenovirus-mediated NIS gene transfer in HepG2 xenografts administration of a therapeutic dose of (131)I or (188)Re (55.5 MBq) resulted in a significant delay in tumor growth and improved survival without a significant difference between (188)Re and (131)I. In conclusion, a therapeutic effect of (131)I and (188)Re was demonstrated in HepG2 xenografts after tumor-specific adenovirus-mediated in vivo NIS gene transfer.