BACKGROUND: Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE: To dynamically measure erythropoietin and hypoxia-inducible factor-1α (HIF-1α) mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING: The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS: Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG (Boster, China), rabbit anti-rat erythropoietin monoclonal antibody (Santa Cruz Biotechnology, USA), and one-step RT-PCR kit (Qiagen, Germany) were used in this study. METHODS: A total of 99 healthy, male, Wistar rats were randomly assigned to three groups: sham surgery (n = 9), non-ischemic preconditioning (n = 45), and ischemic preconditioning (n = 45). In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES: HIF-1α and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS: (1) Intergroup comparison: compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1, 3, and 7 days following reperfusion in the ischemic preconditioning group (P < 0.05 or P < 0.01). Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group (P < 0.05). (2) Temporal expression: HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. (3) Correlation: erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression (r = 0.737, P < 0.01). CONCLUSION: Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemia- induced increase in endogenous HIF-1α expression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.