Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...