Gamma irradiation of red cell concentrates (RCCs) is regularly used to prevent transfusion-associated graft-versus-host disease (TA-GvHD) in at-risk patients. While studies have indicated that irradiated RCCs exhibit increased hemolysis, there have been no efforts to differentiate between free- and microvesicle (MV)-bound hemoglobin (Hb). As an increase in the proportion of free-Hb in irradiated RCCs could alter vascular function, we sought to characterize differences in the state of extracellular Hb based on the timing of irradiation. Four separate pools of seven CPD/SAGM leukoreduced RCCs were produced and split into four sets of seven identical units. The units from each set were subject to irradiation (25 Gy) at six different points during storage, with one unit serving as a nonirradiated control. All testing was performed immediately following unit expiry on day 43. The earlier in storage that units were irradiated, the higher the hemolysis and the lower the proportion of MV-bound Hb. Units irradiated earlier in storage (1-8 days post collection) additionally had lower membrane rigidity (KEI ), lower mean corpuscular Hb concentrations (MCHC), and higher mean corpuscular fragility (MCF). Morphology indices, mean cell volume (MCV), mean corpuscular Hb (MCH), phosphatidylserine (PS) expression, as well as MV production and size did not however differ significantly between groups based on the timing of irradiation. Our findings indicate that irradiation timing can alter the state of extracellular Hb, with "early" irradiation promoting an increased proportion of cell-free Hb as well as mechanical damage to the RBC membrane.