The vasorelaxant properties of red blood cells (RBCs) have been implicated in both the control of normal vascular tone and the protection of tissues from ischemic events. The identity of the vasorelaxant released from RBCs has yet to be elucidated, however growing evidence suggests that nitric oxide bound to the beta 93 cysteine residue of haemoglobin (SNO-Hb) may be responsible. The vasorelaxant moiety is released during the transition of haemoglobin from its R (oxygenated) to T (deoxygenated) state. We subsequently chose to assess the significance of haemoglobin saturation on the capacity of RBCs to mediate hypoxic vasorelaxation. Human RBC samples suspended in saline were manipulated in a thin film rotating tonometer, designed to rapidly change haemoglobin saturation within the time frame of circulatory transit. Various cycles of oxygenation and deoxygenation were performed. The vasorelaxant properties of the RBCs were analysed using an aortic ring bioactivity assay, wherein changes in isometric tension were recorded to study vessel relaxation. The rabbit aortic rings were preconstricted with phenylephrine under hypoxic conditions (approximately 1% O2) prior to RBC addition. Highly saturated RBCs (98.22% +/- 0.45 HbO2) elicited significantly (P<0.001) more relaxation of hypoxic blood vessels compared to those partially saturated (20.40% +/- 5.28 HbO2). Upon re-oxygenation, previously de-oxygenated RBCs were also capable of eliciting vessel relaxation, which was not significantly different from that observed with the original oxygenated RBC relaxation response. Interestingly, the relaxant capability was not simply returned from extracellular milieu upon re-oxygenation. This data provides further evidence that the conformational switch of haemoglobin from the R-state (oxygenated) to the T-state (deoxygenated) is essential for the release of the vasoactive moiety contained within red blood cells.