Recent reports suggest the expression of aquaporin (AQP)-type water channels in mitochondria from liver (AQP8) (Calamita, G., Ferri, D., Gena, P., Liquori, G. E., Cavalier, A., Thomas, D., and Svelto, M. (2005) J. Biol. Chem. 280, 17149-17153) and brain (AQP9) (Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, E., Torgner, I. A., and Ottersen, O. P. (2005) FASEB J. 19, 1459-1467), where they were speculated to be involved in metabolism, apoptosis, and Parkinson disease. Here, we systematically examined the functional consequence of AQP expression in mitochondria by measurement of water and glycerol permeabilities in mitochondrial membrane preparations from rat brain, liver, and kidney and from wild-type versus knock-out mice deficient in AQPs -1, -4, or -8. Osmotic water permeability, measured by stopped-flow light scattering, was similar in all mitochondrial preparations, with a permeability coefficient P(f) approximately 0.009 cm/s. Glycerol permeability was also similar ( approximately 5 x 10(-6) cm/s) in the various preparations. HgCl(2) slowed osmotic equilibration comparably in mitochondria from wild-type and AQP-deficient mice, although the slowing was explained by altered mitochondrial size rather than reduced P(f). Immunoblot analysis of mouse liver mitochondria failed to detect AQP8 expression, with liver homogenates from wild-type/AQP8 null mice as positive/negative controls. Our results provide evidence against functionally significant AQP expression in mitochondria, which is consistent with the high mitochondrial surface-to-volume ratio producing millisecond osmotic equilibration, even when intrinsic membrane water permeability is not high.