1. The permeability of isolated heart mitochondria to mannitol and several other solutes has been examined using a dual isotope procedure. Mannitol enters a portion of the water volume of a centrifuged pellet of mitochondria strictly as a function of its concentration in the suspending medium. Sucrose, KCl, and a number of other solutes penetrate the mitochondrion to the same extent as does mannitol, but high molecular weight solutes such as dextran penetrate a considerably smaller volume. Certain permeant solutes such as glycerol and acetate penetrate to a greater extent than does mannitol. 2. The absolute and relative sizes of the mannitol-permeable and mannitol-impermeable volumes can be varied extensively as a function of the suspending medium. Hypotonic swelling and osmotic swelling which results from either energy-linked ion uptake or gramicidin-induced passive ion uptake cause large increases in the mannitol-impermeable space of the mitochondrion. 3. The size of the mannitol-permeable space does not increase as a function of time in either isotonic or hypotonic media. These results are compatible with earlier suggestions that mitochondria contain two aqueous compartments, one permeable to solutes such as mannitol and sucrose and one which excludes these solutes. In addition, the osmotic barrier to mannitol penetration appears to be the site of both active and passive salt uptake. © 1969.