Cotransfer of thyroid-specific transcription factor (TTF)-1 and Pax-8 gene to tumor cells, resulting in the re-expression of iodide metabolism-associated proteins, such as sodium iodide symporter (NIS), thyroglobulin (Tg), thyroperoxidase (TPO), offers the possibility of radioiodine therapy to non-iodide-concentrating tumor because the expression of iodide metabolism-associated proteins in thyroid are mediated by the thyroid transcription factor TTF-1 and Pax-8. The human TTF-1 and Pax-8 gene were transducted into the human thyroid carcinoma (K1 and F133) cells by the recombinant adenovirus, AdTTF-1 and AdPax-8. Re-expression of NIS mRNA and protein, but not TPO and Tg mRNA and protein, was detected in AdTTF-1-infected F133 cells, following with increasing radioiodine uptake (6.1-7.4 times), scarcely iodide organification and rapid iodide efflux (t(1/2) ≈ 8-min in vitro, t(1/2) ≈ 4.7-h in vivo). On contrast, all of the re-expression of NIS, TPO and Tg mRNA and proteins were detected in F133 cells coinfected with AdTTF-1 and AdPax-8. AdTTF-1- and AdPax-8-coinfected K1 and F133 cells could effectively accumulate radioiodine (6.6-7.5 times) and obviously retarded radioiodine retention (t(1/2) ≈ 25-30-min in vitro, t(1/2) ≈ 12-h in vivo) (P<0.05). Accordingly, the effect of radioiodine therapy of TTF-1 and Pax-8 cotransducted K1 and F133 cells (21-25% survival rate in vitro) was better than that of TTF-1-transducted cells (40% survival rate in vitro) (P<0.05). These results indicate that single TTF-1 gene transfer may have limited efficacy of radioiodine therapy because of rapid radioiodine efflux. The cotransduction of TTF-1 and Pax-8 gene, with resulting NIS-mediated radioiodine accumulation and TPO and Tg-mediated radioiodine organification and intracellular retention, may lead to effective radioiodine therapy of thyroid carcinoma.