The effects of erythrocyte aggregation on O₂release were examined using O₂-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 μm; outer diameter, 100 μm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O₂release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O₂release from erythrocytes in the tube was discussed in relation to the O₂diffusion process. We conclude that the diffusion of O₂from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.