A phospholipid vesicle encapsulating a concentrated hemoglobin solution and pyridoxal 5'-phosphate as an allosteric effector (Hb-vesicle, HbV: diameter, 250 nm) has been developed to provide an O₂ carrying ability to plasma expanders. The O₂ release from flowing HbV was examined using an O₂ permeable fluorinated ethylenepropylene copolymer tube (inner diameter, 28 µm) exposed to a deoxygenated environment. Measurement of the O₂ release was performed using an apparatus that consisted of an inverted microscope, a scanning-grating spectrophotometer with a photon count detector, and the rate of O₂ release was determined based on the visible absorption spectrum in the Q band of Hb. HbV and fresh human RBC were mixed in various volume ratios at [Hb] = 10 g/dL in isotonic saline containing 5 g/dL albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBC were also tested. Since HbV was homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O₂ release from the HbV-RBC mixtures was similar with that from RBC alone. On the other hand, the addition of 50 vol% acellular Hb solution to RBC significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of the O₂ release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O₂ transport.