The norepinephrine transporter (NET) has recently been suggested as a useful reporter gene. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked hNET-green fluorescent protein (GFP) hybrid reporter gene for both nuclear and optical imaging. Methods: A retroviral vector pQCXhNET-IRES-GFP was constructed and used to generate several reporter cell lines and xenografts. Transduced cells were sorted by fluorescence-activated cell sorting based on GFP expression and used for both in vitro and in vivo imaging studies. Results: The transduced reporter cells accumulated 123I- or 124I-labeled metaiodobenzylguanidine (MIBG) to high levels compared with the wild-type parent cell lines. Differences in MIBG accumulation between cell lines were primarily due to differences in influx (K1) rather than efflux (k2). The estimated MIBG distribution volumes (Vd) for transduced Jurkat, C6, and COS-7 cells were 572 ± 13, 754 ± 25, and 1,556 ± 38 mL/g, respectively. A correlation between radiotracer accumulation (K1) and GFP fluorescence intensity was also demonstrated. Sequential imaging studies of mice bearing pQCXhNET-IRES-GFP transduced and wild-type C6 xenografts demonstrated several advantages of 124I-MIBG small-animal PET compared with 123I-MIBG γ-camera/SPECT. This was primarily due to the longer half-life of 124I and to the retention and slow clearance (half-time, 63 ± 6 h) of MIBG from transduced xenografts compared with that from wild-type xenografts (half-time, 12 ± 1 h) and other organs (half-time, 2.6–21 h). Very high radioactivity ratios were observed at later imaging times; at 73 h after 124I-MIBG injection, the C6/hNET-IRES-GFP xenograft-to-muscle ratio was 293 ± 48 whereas the C6 xenograft-to-muscle ratio was 0.71 ± 0.19. Conclusion: These studies demonstrate the potential for a wider application of hNET reporter imaging and the future translation to patient studies using radiopharmaceuticals that are currently available for both SPECT and PET.