1. Changes in the water and ion contents of rabbit renal cortical slices, which had been bathed, immediately after slicing, in air-equilibrated media at room temperature ('freshly prepared slices'), were followed during subsequent incubation at 25 degrees C in oxygenated media of the same composition as the initial bathing medium. 2. In comparison with conventional 'equilibrated' slices (slices incubated at 25 degrees C in oxygenated ordinary medium immediately after slicing) these 'freshly prepared' slices had increased tissue water, sodium and chloride contents and low tissue potassium contents. 3. Control freshly prepared slices incubated at 25 degrees C in oxygenated ordinary medium recovered within 4 min to the tissue water content that is usual for rabbit renal cortical slices incubated in oxygenated ordinary medium at 25 degrees C. Freshly prepared slices incubated at 25 degrees C in oxygenated media containing 1 mM-oubain took 75 min or more to recover to this usual tissue water content. Thus the presence of 1 mM-oubain in both bathing and incubation media produced a marked inhibition of the volume recovery observed when freshly prepared slices are incubated in oxygenated media at 25 degrees C. 4. Reduction of the ouabain concentration reduced the inhibition of cell volume recovery. 5. Replacement of medium glucose by 3-O-methylglucose did not inhibit cell volume recovery in the absence of ouabain. 6. The oxygen consumptions of slices that were bathed and incubated in 1 mM-ouabain media were similar to those of slices initially bathed and incubated in ouabain-free media and then incubated in ouabain media. Thus the effect of ouabain in inhibiting cell volume recovery was unlikely to be secondary to inhibition of cellular energy production. 7. The tissue potassium content of slices incubated aerobically in 1 or 10 mM ouabin fell to an apparently stable value of approximately 100 m-mole/kg dry wt., which corresponds to a calculated concentration ratio of 10:1 across the cellular membrane, suggesting that some residual potassium uptake may still have been occurring. 8. These results indicate that in freshly prepared rabbit renal cortical slices ouabain-sensitive mechanisms play a major role in cell volume recovery. They are not in accord with the postulate that renal cortical cells possess a separate ouabain-insensitive mechanism regulating cell volume.