Isoflurane preconditioning (IsoPC) neuroprotection in experimental stroke is male-specific. We determined whether estradiol alters ischemic outcomes in IsoPC brain and examined the role of estrogen receptors (ERs). Seven to 10 days before preconditioning, ovariectomized (OVX) mice were implanted with estradiol, vehicle, or ER subtype agonists. OVX+/-estradiol, OVX+/-vehicle, OVX+/-ER agonists, and ER subtype wild-type (WT) and knockout (KO) mice were preconditioned for 4 h with sham anesthetic preconditioning (sham PC) or 1% IsoPC and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride staining, with comparisons between IsoPC and corresponding sham PC for each treatment group. Decreased infarct injury was seen in IsoPC OVX+/-vehicle, whereas estradiol in IsoPC OVX mice enhanced ischemic damage. In ER studies, increased infarct volumes were seen in IsoPC ERWT mice regardless of ER subtype. IsoPC in ERalphaKO mice had no effect on infarction volume but reduced only cortical ischemic damage in ERbetaKO mice. In OVX+ERalpha agonist, IsoPC had no effect on infarction volume. In OVX+ERbeta agonist, IsoPC increased cortical infarct volume. Estradiol depresses the brain's protective response to IsoPC and may exacerbate cortical ischemic injury mainly through an ERbeta-dependent mechanism.