BACKGROUND. We evaluated the feasibility of noninvasive micro-single photon emission computed tomography (SPECT)/computed tomography (CT) imaging and quantification of cardiac gene expression after sodium iodide symporter (hNIS) gene transfer in cardiac transplantation. METHODS. Donor rat hearts were perfused ex vivo with adenovirus expressing hNIS (Ad-hNIS), Ad-Null, or University of Wisconsin (UW) solution prior to heterotopic transplantation into syngeneic recipients. In the first group of recipients, imaging of the transplanted hearts with micro-SPECT/CT on day 5 was followed by immediate explant of the organs for ex vivo analyses. Radioactivity counts in the explanted hearts were obtained ex vivo and expressed as a percentage of the injected dose per gram of tissue (%ID/g). Intensities of the SPECT images of the transplanted hearts were quantified and converted to radioactive counts using a standard equation. The second group of recipients was imaged sequentially after injection of I on days 2 to 14 after transplantation. RESULTS. Higher ex vivo radioiodine counts were noted in the hearts perfused with Ad-hNIS (1.04±0.2) compared to either the UW group (0.31±0.11, P<0.001) or the Ad-Null group (0.32±0.08, P<0.001). Image intensity in the Ad-NIS group (0.9±0.2) was also significantly higher than in the UW group (0.4±.03, P=0.003) or the Ad-Null group (0.5±0.1, P<0.05). Sequential imaging of Ad-NIS-perfused hearts between postoperative days 2 and 14 revealed peak image intensity at day 5. Overall, image intensities correlated with ex vivo counts of radioactivity (ρ=0.74, P<0.05). CONCLUSIONS. These data demonstrate that hNIS gene transfer permits sequential real-time detection and quantification of reporter gene expression in the transplanted heart with micro-SPECT/CT imaging. © 2007 Lippincott Williams & Wilkins, Inc.