Glucose transport across the membrane of rat liver microsomal vesicles was studied by a rapid filtration method in three different experimental systems: (i) inward transport in the presence of extravesicular glucose, (ii) efflux from passively preloaded vesicles, and (iii) efflux of glucose generated intravesicularly by glucose-6-phosphatase upon addition of glucose 6-phosphate were investigated. The apparent intravesicular glucose space estimated with the rapid filtration method was lower than the total microsomal glucose accessible space both the in the steady-state phase of uptake and at the starting point of efflux: 0.5 versus 2.3 microl/mg protein. The initial rate of influx/efflux was dependent on the extravesicular/intravesicular glucose concentration and was much lower than the rate of influx estimated previously by the light-scattering technique. Both influx and efflux could be inhibited by N-ethylmaleimide and possibly became saturable at high (>100 mM) glucose concentration. Known inhibitors of GLUT transporters (genistein, cytochalasin B, phloretin, and hexoses) did not affect glucose influx. The time course of glucose efflux from vesicles preincubated in the presence of glucose 6-phosphate was similar to that from glucose-loaded vesicles. These data together with that obtained previously (by a light-scattering technique; Marcolongo, P., Fulceri, R., Giunti, R., Burchell, A., and Benedetti, A. (1996) Biochem. Biophys. Res. Commun. 219, 916-922) indicate that microsomal vesicles are heterogeneous regarding their glucose-transporting properties and that glucose transport is bidirectional and its feature meets the requirements of a facilitative transport.