Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Delta45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Delta45HSV1-tk/GFP/luciferase (Delta45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (~130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Delta45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Delta45-TGL cells compared to nontransduced control cells. The Ki of (14)C-FIAU was 0.49+/-0.02, 0.51+/-0.03, and 0.003+/-0.001 ml/min/g in U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/ nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [(131)I]FIAU (7.4 MBq/animal) or [(124)I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity in the wild-type xenografts. Tissue sampling yielded values of 0.47%+/-0.08%, 0.86%+/-0.06%, and 0.03%+/-0.01%dose/g [(131)I]FIAU in U87-NES-TGL, U87-Delta45-TGL, and U87 xenografts, respectively. The TGL triple-fusion reporter gene preserves the functional activity of its subunits and is very effective for multimodality imaging. It provides for the seamless transition from fluorescence microscopy and FACS to whole-body bioluminescence imaging, to nuclear (PET, SPET, gamma camera) imaging, and back to in situ fluorescence image analysis.