For brown adipose tissue (BAT) to be effective at consuming calories, its blood flow must increase enough to provide sufficient glucose and oxygen to sustain energy expenditure. However, there is no straightforward, noninvasive approach to measure BAT blood flow. We hypothesized that 99mTc-sestaMIBI, a perfusion mediated radiotracer, could provide information about BAT function. We first proved that sestaMIBI could be useful in humans: subcutaneous white fat and deeper sestaMIBI-avid neck fat were collected during surgery and analyzed via microscopy and qPCR. Compared with sc fat, sestaMIBI-avid fat had 300x greater tissue-specific UCP1 expression levels (P<0.001). Cells displayed classic multilocular lipid droplets, polygonal shape, and a high density of mitochondria, consistent with BAT. We next determined how BAT blood flow changes during pharmacological activation. Male 129SVE mice were treated with the ?3-adrenergic receptor agonist CL-316,243 (CL) (1mg/kg) or saline, and then given either 99mTc-sestaMIBI or 18F-FDG, a radiotracer that reflects glucose uptake. CL increased heart rate increased by 23% and respiratory rate by 85% (P<0.001). Tissue perfusion in BAT increased by 61% (P<0.01), with no significant changes in WAT, muscle, liver, or heart. CL increased glucose uptake in BAT by 440% (P<0.01), more than a 10-fold increase compared to WAT and muscle. In summary, our data are consistent with a model in which pharmacologic activation of BAT requires increased blood flow to deliver glucose and oxygen for thermogenesis. However, the glucose consumption exceeds the cardiovascular response, demonstrating that activating BAT could be a safe method to treat obesity and regulate glucose.