1. To trace the early development of the extensive functional membrane abnormalities found in sickle cell anaemia red cells which result from polymerization of haemoglobin S, we followed the effects on Ca and K transport of an in vitro sickling pulse in sickle cell trait (SA) red cells, whose membranes are initially normal.2. Sickling induced a progressively larger uptake of Ca in fed, starved and ATP-depleted SA cells, always substantially higher than that in normal (AA) red cells under comparable conditions. The fraction of ionized Ca within the SA cells, estimated from the equilibrium distribution of (45)Ca induced by the ionophore A23187 was about 0.4 of the total Ca content and similar in SA and AA cells.3. With ATP-depleted SA cells, Ca uptake (representing Ca permeability) was maximal during sickling and was only partially reduced towards normal after desickling. Net Ca uptake during sickling of fed or starved SA cells reverted to net Ca loss upon reoxygenation, irrespective of the Ca gradient, indicating full restoration of the low Ca permeability of the control conditions.4. Following desickling of both fed and starved SA cells, the rates of uphill extrusion of Ca gained during sickling were much smaller than those expected with normal Ca pumps operating at similar internal Ca concentrations.5. After 2 hr sickling ATP levels in starved SA cells were reduced by 50% regardless of the presence or absence of Ca in the medium; therefore sickling-induced Ca uptake was associated with no measurable consumption of ATP due to Ca-pump activity.6. With ATP-depleted SA cells, a Ca uptake of 2-3 mumole/l. cells elicited a maximal response of the K permeability system resulting in full equilibration of the K pools in the cell suspensions. Sickling of fed and starved SA cells produced a small increase in K permeability which was entirely independent of the presence or absence of Ca.7. Sickled forms persisted after reoxygenation only with ATP-depleted SA cells and were more frequent after sickling in the presence of Ca (about 20%) than in a Ca-free medium (about 4%).8. These findings show that initial sickling produces an increased Ca permeability whose extent and reversibility depends on the metabolic state of the cells, and a partial Ca-pump failure, which appears to be irreversible. We confirm a small sickling-related, reversible increase in K permeability but a Ca-dependent increase in K permeability does not occur unless the cells are fully depleted of ATP. The implications for sequential development of related abnormalities in SS cells are discussed.