Discontinuous Stractan gradients were used to separate heterogeneous populations of sickle cells into discrete subpopulations containing varying proportions of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISC). The improved homogeneity of these preparations, together with an enhanced yield of ISC, allowed us to distinguish effects of cell maturation from those of irreversible sickling. With these cell preparations we have begun to define physical properties of ISC. We confirmed the marked abnormalities in cation composition of native ISC. Measurements of ATP in ISC did not substantiate prior reports of ATP deficits. Finally, no evidence for substantial loss of membrane lipids during the process of ISC formation could be demonstrated.