PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 × 106 to 5 × 108 plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m (99mTc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2′-deoxy-2′-fluoro-βD-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 (125l)-labeled FIAU was used in vitro and iodine 131 (131l)-labeled FIAU, in vivo. The 99mTc-labeled P2045 and 125l- or 131l-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. RESULTS: Infected cells in culture trapped 125l-labeled FIAU and 99mTc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% ± 0.4 (mean ± SD) of the added 99mTc-labeled P2045 was trapped, which is significantly lower (P < .05) than the 40% ± 2 of 125l-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 × 108 pfu), the uptake of 99mTc-labeled P2045 was 11.1 % ± 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P < .05) than the uptake of 131l-labeled FIAU at 1.6% ± 0.4 dose per gram. 99mTc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of 99mTc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; 131l-labeled FIAU tumor uptake did not correlate with vector dose. CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector. © RSNA, 2002.