The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-f low electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of >4-to 5-fold and reduced contamination by other cellular membranes. For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-f low light scattering. Osmotic water permeability coefficients (P f ) of 6.1 ؎ 0.2 and 7.6 ؎ 0.9 m⅐s ؊1 were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (E a ; 13.7 ؎ 1.0 and 13.4 ؎ 1.4 kcal⅐mol ؊1 , respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix. In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold higher P f of 690 ؎ 35 m⅐s ؊1 , (ii) a reduced E a of 2.5 ؎ 1.3 kcal⅐mol ؊1 , and (iii) a reversible inhibition by mercuric chloride, up to 83% at 1 mM. These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane. A high TP P f suggests a role for the vacuole in buffering osmotic f luctuations occurring in the cytoplasm. Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.In plants, the cell wall continuum and the cell-to-cell communications (plasmodesmata) provide privileged paths for water exchange and equilibration. Cellular membranes also critically control water transport in a variety of functions, including cell volume and turgor regulation and long-distance transport in nonvascular tissues (1). These functions involve the ability of membranes to transport or sequester osmotic solutes as well as their intrinsic permeability to water (1, 2). The presence in most plant cells of a large vacuole suggests that intra-and transcellular water exchange may depend on the permeability of two membranes in series, the plasma membrane (PM) and the vacuolar membrane or tonoplast (TP) (3, 4). Pressure probe measurements have brought about a better understanding of water relations in a variety of higher plant cell types (1, 5). However, this technique only provides access to the overall cell hydraulic conductivity that includes the water permeabilities of the PM and the TP plus those of the cell wall and the plasmodesmata. The individual water permeabilities of the two membranes thus remain to be determined in these cells (6).The possible existence of water-transporting pores or water channels in membranes of higher plants was discussed nearly 40 years ago (7,8). However, experimental approaches to the molecular mechanisms of membrane water permeability in these organisms h...