The sodium iodide symporter (NIS) is a plasma membrane protein that mediates active uptake of inorganic iodide from plasma into thyroid cells. Expression of the NIS gene in tumor cells may provide a novel mechanism for treating cancer. Previously, we cloned cDNA of rat NIS into the retroviral vector LXSN, transduced human and murine tumor cells, and demonstrated 50-60% killing effect of 131I in cells expressing NIS. However, monolayer cultures of cancer cells cannot adequately represent some aspects of the growth and microenvironmental conditions of three-dimensional (3D) solid tumors. On the other hand, animal models are time consuming and costly and not always reasonable for the first estimation of scientific approaches. An in vitro model of multicellular tumor spheroids growing as a 3D-structure provides an important link between monolayer cell cultures and animal experiments. This model is especially crucial for suicide systems utilizing radioisotopes as the killing agent because the deposition of radiation energy causes DNA damage in cells more effectively in 3D-structure. In this study we used a retroviral vector for introduction of the rat NIS gene into DU145 prostate cancer cells. The liquid-overlay technique was exploited to create a tumor spheroid model. We established that uptake of 125I in monolayer culture cells containing the NIS was 40- to 50-fold greater than in DU145 cells without NIS. Using a clonogenic assay for monolayer culture, we demonstrated a 50-70% killing effect of 131I on DU145 cells expressing the NIS gene. The same dose of 131I resulted in complete death of tumor spheroids composed of the DU145-NIS cells. Our data demonstrates significant anti-tumor efficacy of novel radioisotope concentrator gene therapy in a multicellular spheroid model, thus proving to be a useful link between the in vitro system and in vivo animal model.