The identification of molecular water transporters and the generation of transgenic mice lacking water transporting proteins has created a need for accurate methods to measure water permeability. This review is focused on methodology to characterize water permeability in living cells and complex multicellular tissues. The utility of various parameters defining water transport is critically evaluated, including osmotic water permeability (P(f)), diffusional water permeability (P(d)), Arrhenius activation energies (E(a)), and solute reflection coefficients (sigma(p)). Measurements in cellular and complex tissues can be particularly challenging because of uncertainties in barrier geometry and surface area, heterogeneity in membrane transporting properties, and unstirred layer effects. Strategies to measure plasma membrane P(f) in cell layers are described involving light scattering, total internal reflection fluorescence microscopy, confocal microscopy, interferometry, spatial filtering microscopy, and volume-sensitive fluorescent indicators. Dye dilution and fluorescent indicator methods are reviewed for measurement of P(f) across cell and tissue barriers. Novel fluorescence and gravimetric methods are described to quantify microvascular and epithelial water permeabilities in intact organs, using as an example lungs from aquaporin knockout mice. Finally, new measurement strategies and applications are proposed, including high-throughput screening for identification of aquaporin inhibitors.