Over the past twenty years, studies investigating sickle cell pathophysiology have primarily focused on the dehydration process. Although this has lead to some significant insight into the sickling process and new therapeutic treatments, we still lack a fundamental understanding of the disease. Recent in vitro and in vivo studies have identified older, low-density (ρ<1.087 g/cc) sickle red blood cells (SS RBC) that are resistant to dehydration mediated by valinomycin or the Ca-activated K channel. We hypothesize that these light cells most likely derive from dense SS RBC that become rehydrated. To study rehydration, we subjected dense SSRBC (ρ>1.107 g/cc) to either oxygenated incubation or in vitro fast oxy/deoxy cycling using a dual channel, fast cycle apparatus (FCA) to simulate in vivo oxy/deoxy conditions. After oxy incubation or oxy/deoxy cycling, the number of light cells, (ρ<1.087 g/cc), was assessed. There was some light cell generation during static incubation at 37°C (3.0 +/- 2.2%, n=41). Fast oxy/deoxy cycling increased light cell formation (5.4 +/- 2.4%, n=41, p< 10 -11 ). These results support the hypothesis that rehydrated SS RBCs derive from dense cells. The rehydrated cells were K depleted (K + = 20 +/- 14 mmol/KgHb, n=5), Na + loaded (Na + = 394 +/- 106 mmol/KgHb, n=12), and had high levels of externalized phosphatidylserine, particularly in the presence of calcium. The presence of 1.5 mM external Ca ++ inhibited the formation of rehydrated SS RBC by 38% (p<0.01, n=7) during oxy/deoxy cycling. This inhibitory Ca ++ effect was prevented by the presence of 1 μM charybdotoxin (p<0.01, n=5), implying a competing dehydration effect from the Ca-activated K channel. Pre-incubation of the dense SS RBC with 45 μM DIDS, or the presence of 1 mM bumetanide, inhibited the generation of light cells during fast oxy/deoxy cycling by 39% (p<0.004, n=7) and 45% (p<0.002, n=8), respectively. These results support the hypothesis that the sickling-induced pathway, previously implicated in SS RBC dehydration, may be involved in the rehydration of dense, K depleted cells. 1 mM bumetanide also inhibited light cell generation during oxy incubation (p<0.05, n=9) suggesting a novel, bumetanide sensitive pathway in SS RBC rehydration.