A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible approximately 12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 +/- 0.05 s at 23 degrees C, corresponding to an osmotic water permeability (Pf) of approximately 0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 +/- 0.5 vs. 5.5 +/- 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.