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A new method has been developed for measuring the permeability coefficient, w, of small nonelectrolytes. The method depends upon a mathematical analysis of the time course of cell volume changes in the neighborhood of the minimum volume following addition of a permeating solute to an isosmolal buffer. Coefficients determined by the minimum volume method agree with those obtained using radioactive tracers. w for urea in human red cells was found to decrease as the volume flow, J,, into the cell increased. Such behavior is entirely unexpected for a single uniform rate-limiting barrier on the basis of the linear phenomenological equations derived from irreversible thermodynamics. However, the present findings are consonant with a complex membrane system consisting of a tight barrier on the outer face of the human red cell membrane and a somewhat less restrictive barrier behind it closer to the inner membrane face. A theoretical analysis of such a series model has been made which makes predictions consistent with the experimental findings.When red cells are placed in a medium containing an isosmolal concentration of impermeant solute, together with a suitable concentration of permeant solute, the cell volume initially shrinks and then returns to its initial volume, after passing through a clearly defined minimum. In 1933 Jacobs (1) had already pointed out that the minimum volume and the time taken to achieve it could be used to determine the permeability coefficient of the permeant solute; the present method is based upon this suggestion. In the shrinking phase water moves out of the red cell because of the osmotic pressure gradient due to the addition of the permeant solute to the medium. At the same time the permeant solute diffuses into the cell down its concentration gradient.

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