Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.