O₂ transport and O₂ diffusion interact in providing O₂ to tissue, but the extent to which diffusion may be critical in the heart is unclear. If O₂ diffusion limits mitochondrial oxygenation, a change in blood O₂ affinity at constant total O₂ transport should alter cardiac O₂ consumption (O₂) and function. To test this hypothesis, we perfused isolated isovolumically working rabbit hearts with erythrocytes at physiological blood-gas values and P₅₀ (PO₂ required to half-saturate hemoglobin) values at pH of 7.4 of 17 ± 1 Torr (2,3-bisphosphoglycerate depletion) and 33 ± 5 Torr (inositol hexaphosphate incorporation). When perfused at 40 and 20% of normal coronary flow, mean O₂ decreased from the control value by 37 and 46% (P < 0.001), and function, expressed as cardiac work, decreased by 38 and 52%, respectively (P < 0.001). Perfusion at higher P₅₀ during low-flow ischemia improved O₂ by 20% (P < 0.001) and function by 36% (P < 0.02). There was also modest improvement at basal flow (P < 0.02 and P < 0.002, respectively). The improvement in O₂ and function due to the P₅₀ increase demonstrates the importance of O₂ diffusion in this cardiac ischemia model.