The aging of human erythrocytes stored in vitro at 4 degrees C was studied by the entrapping method. Erythrocytes were subjected to a sudden hyposmolar stress by suspension in solutions of varying osmolarity in the presence of the spin label tempocholine. The curves obtained when the amount of the spin label entrapped in the ghosts after resealing was plotted against the osmolarity of the buffer solutions exhibited an entrapping which increased with time of the in vitro storage of the erythrocytes (40-60 and 80-100 days). Rejuvenation of the aged erythrocytes by addition of glucose-adenine restored the entrapping curves to shapes nearly similar to those obtained with erythrocytes collected 30-35 days earlier.