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Erythrocyte ATP depletion during storage of ACD blood is associated with disc-sphere transformation and a decrease in membrane viscoelastic properties. A heterogeneity within the stored cells is indicated by the presence of both biconcave and spherical cells and by bimodal derivative curves of osmotic fragility.The heterogeneity in size of the ghosts, prepared from red cells at various times of storage from ACD collected blood and its dependence on erythrocyte ATP and calcium content was studied by multichannel particle size analysis. Reconstituted ghosts were prepared by osmotic hemolysis followed by restoration of isotonicity. A single population of ghosts was obtained when fresh red cells were used. ATP depletion during blood storage was accompanied by a bimodal size distribution of erythrocyte ghosts due to the appearance of a second ghost population of smaller size. Such bimodality was not observed in the size distribution of intact stored erythrocytes. The population of smaller ghosts increased in number during prolonged storage while the normal population proportionally decreased. After 7 t o 8 weeks storage of red cells, about 50% of resultant ghosts were represented by a second population. A similar population was produced from fresh cells when CaCl, (1 x 1 0 -6~) was introduced into ghosts during hemolysis.The introduction of nucleotides (ATP, ITP, GTP, ADP, AMP) or EDTA (14 x 1 0 -s~) into ghosts during hemolysis of stored cells prevented the accumulation of small ghosts. The inhibitory effect of nucleotides was greater than predicted from stability constants for C a t + indicating their specific interaction with the membrane.Determination of the calcium content in extracts of dry ashed red cells by atomic absorption spectrophotometry revealed an increase during prolonged blood storage from 11.3f6.3 ,ug atoms/l cells (first two weeks of storage) to 22.0h7.0 (7-9 weeks of storage). The calcium content of both fresh and stored cells was quantitatively recovered from the ghosts.Kinetic data revealed that upon restoration of isotonicity, ghosts became smaller in volume than the corresponding erythrocytes but subsequently swelled to a final stable volume. The second ghost population underwent markedly little reswelling. This is indicative of increased membrane rigidity which prevents changes in volume, and decreased passive permeability to NaCl.