The dimensions of the aqueous pore in aquaporins (AQP) 0, 1, 2, 3, 4, and 5 expressed in Xenopus laevisoocytes were probed by comparing the ability of various solutes to generate osmotic flow. By improved techniques, volume flows were determined from initial rates of changes. Identical values for the osmotic water permeability (L _p) were obtained in swelling as in shrinkage experiments demonstrating, for the first time, that aquaporins are bidirectional. The reflection coefficients (¿) of urea, glycerol, acetamide, and formamide at 23¿°C were: AQP0: 1, 1, 0.8, 0.6; AQP1: 1, 0.8, 1, 1; AQP2: 1, 0.8, 1, 1; AQP3: 1, 0.2, 0.7, 0.4; AQP4: 1, 0.9, 1, 1; and AQP5: 1, 1, 1, 0.8. As seen there is no clear connection between solute size and permeation. At 13¿°C the ¿s for AQP3 were 1, 0.4, 1, and 0.5; functionally, this pore narrows at lower temperatures. HgCl₂ reversibly reduced theL _p of AQP3 and increased ¿_glyc to 1 and ¿_form to 0.6. We conclude that the pore of the various aquaporins are structurally different and that a simple steric model is insufficient to explain solute-pore interactions.