Multidrug resistance-associated protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qualitative assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomography (PET) and a PET probe. 6-Bromo-7-[(11)C]methylpurine was designed to readily enter the brain after intravenous administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[(11)C]methylpurine into mice. The efflux rate of the substrate was kinetically estimated to be 1.4 h(-1) with high precision. Moreover, knockout of Mrp1 gene caused approximately a 90% reduction of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quantitative assessment for MRP1 function in the living brain.