Our objective was to determine whether multiple clinically useful radiotracers accumulate in brown adipose tissue (BAT) and to assess their uptake in rats kept at room temperature or exposed to a cold environment. Methods: The following radiotracers were injected intravenously into groups of 6 female Wistar rats: 201Tl-chloride (TlCl), 123I-metaiodobenzylguanidine (MIBG), 99mTc-sestamibi (MIBI), 18F- or 3H-FDG, 3H-l-methionine, and 3H-thymidine. BAT-stimulated animals were maintained at 4°C for 4 h before tracer injection, whereas control animals were kept at approximately 22.5°C. The animals were sacrificed at 20–60 min after tracer injection, and BAT, major organs, and blood were extracted, weighed, and measured for radioactivity. The localization of uncoupling protein-1, glucose transporter-1, and norepinephrine transporter was evaluated with immunohistochemical staining in both groups. Results: We determined the percentage injected dose (%ID) per gram of each radiotracer in interscapular BAT, normalized to blood %ID/g. In control animals, this uptake ratio (±SD) was 8.44 ± 3.39 for 201TlCl, 9.77 ± 6.06 for 123I-MIBG, 37.30 ± 14.42 for 99mTc-MIBI, 5.47 ± 4.44 for 18F- or 3H-FDG, 1.93 ± 0.87 for 3H-l-methionine, and 1.22 ± 0.74 for 3H-thymidine. Compared with uptake at room temperature, uptake after exposure to cold increased 26.4-fold (P < 0.01) for 18F- or 3H-FDG and increased significantly (P < 0.05) for 201Tl (2.04-fold), 123I-MIBG (3.25-fold), and 3H-l-methionine (3.11-fold). Immunohistochemical staining revealed increased glucose transporter-1 and norepinephrine transporter expression in BAT cell membranes and blood vessels after exposure to cold, whereas uncoupling protein-1 was expressed in the cytoplasm under both control and cold-stimulated conditions. Conclusion: BAT uptake of 18F- or 3H-FDG, 123I-MIBG, and 3H-l-methionine was significantly increased over the control state by exposure to cold. Increased uptake of 201TlCl relative to blood in cold-stimulated BAT suggests that blood flow in BAT is increased by exposure to cold. The greater increased uptake with 18F- or 3H-FDG, 123I-MIBG, and 3H-l-methionine, and the immunohistostaining findings, suggest that other factors in addition to blood flow (e.g., increased metabolism, increased transport, or metabolic trapping of the tracers) are involved in cold-stimulated BAT activation. Knowledge that high uptake in BAT may possibly be observed on clinical scans using several radiotracers, especially after patients are exposed to the cold, may lead to more accurate interpretation of clinical studies.