Morphine is converted to morphine 3-ß-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 µM). Therefore, the build-up of high (about 20 µM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 µM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion.