Objectives: Breathing a hyperoxic gas (≧95% O₂) protects against ischaemia-reperfusion injury in rat and mouse hearts. The present study investigated how oxygen concentration and duration of hyperoxic exposure influenced cardioprotection, and whether hyperoxia might induce delayed cardioprotection (after 24 h). Methods: Animals were kept in normal air or in a hyperoxic environment, and their hearts were isolated and Langendorff-perfused immediately or 24 h thereafter. Global ischaemia was induced for 25 min in rats and 40 min in mice, followed by 60 min of reperfusion. Infarct size was determined by triphenyl tetrazolium chloride staining. Results: In rats exposure to ≧95, 80, and 60%, but not to 40% of oxygen immediately before heart isolation and perfusion improved postischaemic functional recovery. Eighty or more percent of oxygen also reduced infarct size. A preconditioning-like effect could be evoked by 60 or 180 min of hyperoxia, giving both immediate and delayed protection. In the mouse heart protection could be induced by pretreatment for 15 or 30, but not by 60 min with ≧95% oxygen. The protective effect of hyperoxia in mice could be evoked in the immediate model only. Conclusions: Hyperoxia protects the isolated rat and mouse heart against ischaemia-reperfusion injury, but some species-different responses exist. The protection depends on both oxygen concentration in inspired air, and duration of hyperoxic exposure.