Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.