Water and nonelectrolyte permeability in basolateral membrane vesicles (BLMV) isolated from rat and rabbit renal cortex were characterized. Osmotic water (Pf) and solute (Ps) permeabilities were determined from the time course of scattered light intensity in response to osmotic gradients. In rabbit BLMV, Pf = 2.5 X 10(-2) cm/s, Purea = 1.2 X 10(-6) cm/s, and sigma urea = 0.95 (23 degrees C). Relative solute permeabilities were urea, 1; thiourea, 0.83; ethylene glycol, 9.7; glycerol, 4.6; formamide, 20; and acetamide, 12. Pf and Purea were not altered by organic mercurials, phloretin, urea, and high-affinity urea analogues. delta H (12-50 degrees C) was 10.7 and 2.5 kcal/mol for Purea and Pf, respectively; in contrast, reported delta H for Pf in rabbit brush-border membrane vesicles (BBMV) was 2 kcal/mol (T less than 33 degrees C) and 14 kcal/mol (T greater than 33 degrees C). To examine whether membrane fluidity changes were associated with this difference, fluorescence anisotropy decay was measured using 1,6-diphenyl-1,3,5-hexatriene (DPH). In both BLMV and BBMV, DPH rotation was hindered at all temperatures (5-53 degrees C). Changes in steady-state anisotropy were attributable to changes in DPH rotational freedom rather than to changes in DPH rotational rate without evidence for a definitive membrane thermotropic phase transition. These results suggest that BLMV urea transport occurs by lipid diffusion and that osmotic water transport is rapid and may be facilitated. A comparison of transepithelial Pf with BLMV and BBMV Pf is consistent with transcellular osmotic water flow in the proximal tubule.