Sickle cell disease (SCD) is one of the commonest severe inherited disorders with over 350,000 deaths yearly. It is caused by the presence of the abnormal haemoglobin HbS in patients’ red blood cells (RBCs) instead of the normal adult HbA. When deoxygenated, HbS polymerises and distorts RBCs into sickles and other bizarre shapes. Clinical sequelae involve chronic anaemia with acute ischaemic episodes causing pain, organ damage and early death [1]. SCD RBCs have increased cation permeability which results in solute loss, shrinkage and increased [HbS]. On deoxygenation, the lag time to polymerisation is inversely proportional to [HbS]15-30, so modest shrinkage markedly encourages sickling. Three main pathways are involved [2]: KCl cotransport, the Ca2+-activated K+ channel (Gardos channel) and a deoxygenation-induced non-selective cation pathway (Psickle). Psickle has a central role mediating Ca2+ entry, activating the Gardos channel and causing other sequelae such as Ca2+-induced phosphatidylserine exposure. Deoxygenated RBCs from SCD patients also undergo haemolysis in isosmotic solutions of certain non-electrolytes [3]. Lysis shares a number of features in common with Psickle activation, suggesting its involvement. We have used this phenomenon to design a novel diagnostic and prognostic test for SCD which makes use altered RBC permeability rather than presence of HbS per se [4]. Here we further characterise the lysis pathway. RBCs from SCD (HbSS) patients were washed in MOPS-buffered saline (in mM: NaCl 145, glucose 5, MOPS 10, pH 7.4, 290±5mOsm.kg-1, 37oC). The lysis solution was isosmotic sucrose solution (with sucrose replacing NaCl). At pH 7.4, RBCs lyse on deoxygenation. Pre-incubation of RBCs with cytochalasin B (10µM; Figure 1), latrunculin A (5µM) and mycalolide B (5µM) inhibited lysis. IC50 for cytochalasin B was about 1µM. By contrast, incubating RBCs with phalloidin (10µM) increased haemolysis. With all inhibitors, O2 saturation fell to values not significantly different from 0% within 20min (4±3%, mean±SEM, n=3, for cytochalasin B). Reagents targeting actin therefore modulate the lysis pathway activated on deoxygenation of sickle RBCs in isosmotic sucrose. Effects were not due to alteration in O2 saturation of Hb. We hypothesise that inhibiting cytoskeletal actin polymerisation with cytochalasin B, latrunculin A and mycalolide B prevents HbS aggregates from opening the Psickle-like lytic pathway, whilst phalloidin stabilises actin polymers which may increase RBC permeability leading to greater haemolysis. Results are important for understanding the nature of the abnormal permeability of sickle cells, which is a pre-requisite to the rational design of inhibitory compounds to ameliorate the clinical features of SCD.