Estradiol-17beta is released from the ovaries in a cyclic manner during the normal estrous cycle in rats. During the transition from the diestrous to proestrous stage, the 17beta-estradiol increases in blood circulation. We hypothesized that a higher serum level of endogenous 17beta-estradiol would protect hippocampal pyramidal neurons against global cerebral ischemia via activation of the cyclic-AMP response element binding protein (CREB)-mediated signaling cascade. Furthermore, we asked if a single 17beta-estradiol bolus provides protection against ischemia in the absence of endogenous estradiol. To test these hypotheses, rats were subjected to global cerebral ischemia at different stages of the estrous cycle. Ischemia was produced by bilateral carotid occlusion and systemic hypotension. Brains were examined for histopathology at 7 days of reperfusion. Higher serum levels of 17beta-estradiol (at proestrus and estrus stages) correlated with increased immunoreactivity of pCREB in hippocampus and ischemic tolerance. At diestrus, when circulating gonadal hormone concentrations were lowest, the pCREB protein content of hippocampus was reduced and showed the least number of normal neurons after ischemia compared to other stages of the estrous cycle. A similar phosphorylation pattern was also observed for mitogen-activated protein kinase (MAPK) and calcium-calmodulin-dependent protein kinase (CaMKII) in hippocampus. The cyclic variation in ovarian hormones did not reflect phosphorylation of protein kinase B (Akt). To test the efficacy of a single bolus of 17beta-estradiol before ischemia, ovariectomized rats were treated with 17beta-estradiol (5/10/50 microg/kg) or vehicle (oil) and 48/72/96 h later rats were exposed to cerebral ischemia. A single 17beta-estradiol bolus treatment in ovariectomized rats significantly increased CREB mRNA activation and protected CA1 pyramidal neurons against ischemia. These results suggest that an exogenous bolus of 17beta-estradiol to ovariectomized rats protects hippocampus against ischemia via activation of the CREB pathway in a manner similar to the endogenous estrous cycle.