The osmotic water permeability Pf of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations. Pf values for BBM and BLM preparations of small intestine were equal and amount to 60 microns/sec. For renal preparations, Pf values amount to 600 microns/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) microns/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3 +/- 0.6 and in renal membranes 1.0 +/- 0.3 kCal/mole, between 25 and 35 degrees C. The mercurial sulfhydryl reagent pCMBS inhibited completely and reversibly the high Pf value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the high Pf values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to approximately 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.