An ionophore specific for divalent cations has been used to load normal erythrocytes and erythrocytes from patients with sickle cell anaemia (Hb SS disease), with small amounts of calcium. Such calcium accumulation leads to decreased cellular water, potassium, adenosine triphosphate (ATP), and osmotic fragility, all characteristics of irreversibly sickled cells (ISCs). In addition, calcium loading of Hb SS, but not normal, erythrocytes causes a marked decrease of haemoglobin oxygen affinity; another, and specific, hallmark of ISCs. Ionophore-induced accumulation of calcium by deoxygenated Hb SS erythrocytes also leads to temporary retention of sickled shape following reoxygenation, despite the absence of detectable intracellular haemoglobin S fibres. All these effects require calcium in the incubation medium and support the idea that increased intracellular calcium is important in the formation of ISCs in patients with Hb SS disease.