Phosphatidylserine (PS) is usually confined to the inner leaflet of red blood cells (RBCs) through inactivity of a Ca2+-dependent scramblase and high activity of the aminophospholipid translocase (APLT). Exposure occurs in a small, but variable, percentage of RBCs from sickle cell disease (SCD) patients (1). It may contribute to anaemia and ischaemia. PS exposure in RBCs from SCD patients is increased on deoxygenation in the presence of physiological levels of Ca2+o and is prevented by Ca2+i buffering (2). Ca2+ entry is via the deoxygenation-induced cation conductance pathway. Only low micromolar Ca2+i levels are required (2 cf. 3). The mechanism(s) by which Ca2+ has its effect, however, remain(s) unclear. It may be direct via inhibition of APLT and scramblase activation. Alternatively Ca2+ may act indirectly via second messengers, which may involve Gardos channel activation and RBC shrinkage (4). These possibilities have been tested here. Routine discarded blood samples were obtained from HbSS SCD patients, using EDTA as anticoagulant. RBCs were washed into high K+ (HK) Hepes-buffered saline (HBS) comprising (in mM): 90 KCl, 46 NaCl, 10 Hepes, 10 inosine, 0.15 MgCl2 and in final step containing 2mM EGTA to remove contaminant Ca2+. To alter [Ca2+]i RBCs (0.5% Hct) were exposed to the ionophore Br-A23187 (2.5 or 6µM) and different [Ca2+]o clamped with 2mM EGTA in low K+ (LK) (4mM KCl and 132mM NaCl) or HK HBS with 1mM vanadate (30min, 37oC). All solutions were pH 7.4 at 37oC, osmolality 290mOsM. Controls and inhibitors were tested in parallel. PS exposure was assessed using FITC-lactadherin (16nM, 105 RBCs, Haematologic Technologies Inc.) and flow cytometry (2). Incubation in HK saline significantly reduced PS exposure compared with LK HBS (p<0.0001, Figure 1a), using 2.5µM ionophore. When 6µM Br-A23187 was used, the difference between HK and LK was less apparent. Various inhibitors were tested: Z-VAD-FMK (to 60µM), a non-specific caspase inhibitor; quinacrine (to 100µM) a phospholipase A2 inhibitor; ABT491 (to 50µM) a PAF receptor antagonist; diclofenac (to 500µM) and acetylsalicyclic acid (to 200µM), cyclooxygenase inhibitors to prevent PG synthesis; and GW4869 (to 10µM) and 3,4-dichlorocoumarin (to 200µM), sphingomyelinase inhibitors to prevent ceramide production. None of these inhibitors resulted in a significant change in PS exposure (eg diclofenac, Figure 1b). Findings indicate that Ca2+-dependent PS exposure is unlikely to involve PG, PLA2, ceramide or caspases. However, PS exposure was reduced in the absence of an outward K+ gradient. Possible mechanisms include prevention of K+ loss and cell shrinkage, loss of hyperpolarisation or sensitivity to K+o per se. These possibilities are currently being tested.