The dimensions of the aqueous pore in aquaporins (AQP) 0, 1, 2, 3, 4, and 5 expressed in Xenopus laevis oocytes 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 (Lp) were obtained in swelling as in shrinkage experiments demonstrating, for the first time, that aquaporins are bidirectional. The reflection coefficients (sigma) of urea, glycerol, acetamide, and formamide at 23 degreesC 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 degreesC the sigmas for AQP3 were 1, 0.4, 1, and 0.5; functionally, this pore narrows at lower temperatures. HgCl2 reversibly reduced the Lp of AQP3 and increased sigmaglyc to 1 and sigmaform 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.