Purpose: Recently we have successfully tracked the migration and homing of AC133+ progenitor cells (AC133PCs) at the site of neovascularization in subcutaneous as well as in intracranial tumors by in vivo and ex vivo MRI. The tracking was possible due to magnetic labeling of cells using ferumoxides and protamine sulfate (FePro). Accessibility, easy harvesting and established techniques for genetic manipulation renders AC133PCs as attractive cellular vehicles when systemic or local gene delivery is required. The purpose of the study was to investigate the ability of AC133PCs to carry a gene to the tumor sites and use these transgenic cells as cellular probes to track the migration of the cells by magnetic resonance imaging (MRI). Material and Methods: Lentiviral vectors carrying human sodium iodide symporter (hNIS) with different promoters (such as CMV and EF1) were used to optimize the transfection efficiency of AC133PCs. Transgenic cells were also subjected to long term culture to determine the peak transfection time and the stability of transfection. For animal studies, transgenic cells were magnetically labeled using FePro on day four following transfection and the viability of the cells was determined. Mixtures of transgenic and transgenic plus magnetically labeled or non-transgenic magnetically labeled AC133PCs were administered intravenously in rats bearing U251 glioma on day 11 following intracranial implantation of 5x105 U251 cells. MR images were obtained pre and 7 days post administration of the cells to determine the migration of transgenic cells in the tumors. Following MRI, SPECT images were obtained to determine the expression of hNIS in the tumors using Tc-99m-pertechnetate (Tc-99m). Collected brains were analyzed histochemically to determine the iron labeled cells and cells expressing hNIS. Results: Both CMV and EF1 promoters equivocally expressed hNIS in AC133PCs, which was determined by Tc-99m uptake study. MRI images showed low signal intensity areas in tumor that received magnetically labeled cells, which was significantly different from the MRI that was obtained before the administration of cells in the same animal. SPECT images showed increased Tc-99m activity in tumor that received transgenic cells compared to the tumor that received only magnetically labeled cells. Histochemically, both iron and hNIS positive cells were determined in the tumors. Conclusion: Transgenic AC133PCs can be used both as gene carrier/delivery system and probes for cellular MRI.