Since it is not known why sickle RBCs tend to undergo microvesiculation, we have investigated their susceptibility to thermal stress. While normal RBCs start to vesiculate at 49.0 ± 0°C (n = 14), sickle RBCs begin to vesiculate at 47.9 ± 0.5°C with a range of 46,5 to 48,5°C (n = 14). This abnormality is reproduced by treating normal RBCs with phenazine methosulfate (PMS), which stimulates the excess intracellular generation of superoxide characteristic of sickle RBCs. For PMS-treated RBCs, there is a strong correlation between membrane protein thiol oxidation and vesiculation temperature (r = .977, P < .001). The abnormal vesiculation temperature of both unmanipulated sickle RBCs and PMS-treated RBCs is significantly improved by treatment of the RBCs with dithiothreitol. The most dense sickle RBCs are most prone to vesiculation during thermal stress, and they are the subpopulation having the greatest amount of thiol oxidation. We conclude that the tendency of sickle RBCs to vesiculate during thermal stress is further evidence for functional abnormality of the RBC cytoskeleton due to thiol oxidation.