The question of how often erythrocytes should be washed in order to obtain a protein-free and therapeutically efficient unit of washed red blood cells is still controversial. To obtain more information regarding this problem, 6 X 15 units of whole blood were washed 1-6 times with a saline solution immediately and after 7, 14, 21, 28 and 35 days of storage. After each washing procedure the content of total protein, of immunglobulins A, G, and M and of complement factor C3 was determined in the supernatants, as well as the osmotic fragility in the corresponding erythrocyte sediments. The statistically analyzed results enabled us to describe the average degree of influences as equations and to recommend a preparation prescription. The results show that the protein levels decrease precipitously after the first and second washing procedures. The following 4 procedures have no significant effect. On the other hand, with increasing time of storage more washing procedures are necessary to reach the same results than with younger units. That is also true for the concentrations of immunoglobulins and complement C3. The osmotic fragility of the erythrocytes increases with each washing procedure as well as each day of storage. According to these results, an optimal, protein-free and effective unit of washed red blood cells is obtained when a 1-2-week-old unit of whole blood is washed 2-3 times.