Purpose: Both human NIS and mutant D2R transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor (D2R) and compared its characteristics. Materials and Methods: The recombinant plasmid (pIRES-hNIS/D2R) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter. pIRES-hNIS/D2R was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND (SK-Hep1-hNIS/D2R) cells stably expressing hNIS and D2R was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and D2R genes. The expressions of hNIS and D2R were measured by 125I uptake assays and receptor binding assays. Specific binding of D2R to [3H]spiperone was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. Kd and Bmax values were estimated. The correlation between hNIS and D2R expression was compared by using each clone. Results: Similar quantities of hNIS and D2R genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. 125I uptake in HEP-ND cells was completely inhibited by KClO4, a NIS inhibitor. Specific binding to HEP-ND cells was saturable and the Kd and Bmax values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and D2R binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and D2R in a stably transfected cell line. We expect that D2R and hNIS genes can complement mutually as a nuclear reporting system or that D2R can be used as reporter gene when hNIS gene were used as a treatment gene. (Korean J Nucl Med 38(4):294-299, 2004)