Nonspecific quantitation of [14C]sucrose in blood and brain has been routinely used as a quantitative measure of the in vivo blood-brain barrier (BBB) integrity. However, the reported apparent brain uptake clearance (Kin) of the marker varies widely (∼100-fold). We investigated the accuracy of the use of the marker in comparison with a stable isotope of sucrose ([13C]sucrose) measured by a specific liquid chromatography-tandem mass spectrometry method. Rats received single doses of each marker, and the Kin values were determined. Surprisingly, the Kin value of [13C]sucrose was 6- to 7-fold lower than that of [14C]sucrose. Chromatographic fractionation after in vivo administration of [14C]sucrose indicated that the majority of the brain content of radioactivity belonged to compounds other than the intact [14C]sucrose. However, mechanistic studies failed to reveal any substantial metabolism of the marker. The octanol:water partition coefficient of [14C]sucrose was >2-fold higher than that of [13C]sucrose, indicating the presence of lipid-soluble impurities in the [14C]sucrose solution. Our data indicate that [14C]sucrose overestimates the true BBB permeability to sucrose. We suggest that specific quantitation of the stable isotope (13C) of sucrose is a more accurate alternative to the current widespread use of the radioactive sucrose as a BBB marker.