Whole cell deformability of ATP-depleted and Ca-loaded red cells has been measured at various osmolalities to determine those cellular factors responsible for the reduce deformability of these cells. For cells depleted of ATP in Ca-free medium, a progressive loss of hypotonic deformability identified membrane loss with reduced surface area-to-volume ratio as the dominant mechanism of deformability loss. For cells treated with Ca and the ionophore A23187 without prior depletion, a rapid loss of isotonic deformability, reversible in hypotonic medium, identified dehydration with increased internal viscosity as the dominant mechanism of deformability loss. In contrast to previously held concepts, increased membrane rigidity was not found to have a major influence.