Objective: We studied the concordance of transgene expression in the transplanted heart using bicistronic adenoviral vector coding for a transgene of interest (human carcinoembryonic antigen: hCEA - beta human chorionic gonadotropin: βhCG) and for a marker imaging transgene (human sodium iodide symporter: hNIS). Methods: Inbred Lewis rats were used for syngeneic heterotopic cardiac transplantation. Donor rat hearts were perfused ex vivo for 30 min prior to transplantation with University of Wisconsin (UW) solution (n = 3), with 109 pfu/ml of adenovirus expressing hNIS (Ad-NIS; n = 6), hNIS-hCEA (Ad-NIS-CEA; n = 6) and hNIS-βhCG (Ad-NIS-CG; n = 6). On postoperative day (POD) 5, 10, 15 all animals underwent micro-single photon emission computed tomography/computed tomography (SPECT/CT) imaging of the donor hearts after tail vein injection of 1000 μCi 123I and blood sample collection for hCEA and βhCG quantification. Results: Significantly higher image intensity was noted in the hearts perfused with Ad-NIS (1.1 ± 0.2; 0.9 ± 0.07), Ad-NIS-CEA (1.2 ± 0.3; 0.9 ± 0.1) and Ad-NIS-CG (1.1 ± 0.1; 0.9 ± 0.1) compared to UW group (0.44 ± 0.03; 0.47 ± 0.06) on POD 5 and 10 (p < 0.05). Serum levels of hCEA and βhCG increased in animals showing high cardiac 123I uptake, but not in those with lower uptake. Above this threshold, image intensities correlated well with serum levels of hCEA and βhCG (R2 = 0.99 and R2 = 0.96, respectively). Conclusions: These data demonstrate that hNIS is an excellent reporter gene for the transplanted heart. The expression level of hNIS can be accurately and non-invasively monitored by serial radioisotopic SPECT imaging. High concordance has been demonstrated between imaging and soluble marker peptides at the maximum transgene expression on POD 5. © 2007 European Association for Cardio-Thoracic Surgery.