The mechanisms underlying the age-dependent reversal of female cardioprotection are poorly understood and complicated by findings that estrogen replacement is ineffective at reducing cardiovascular mortality in postmenopausal women. Although several protective signals have been identified in young animals, including PKC and Akt, how these signals are affected by age, estrogen deficiency, and ischemia-reperfusion (I/R) remains unknown. To determine the independent and combined effects of age and estrogen deficiency on I/R injury and downstream PKC-Akt signaling, adult and aged female F344 rats (n = 12/age) with ovaries intact or ovariectomy (Ovx) were subjected to I/R using Langendorff perfusion (31-min global-ischemia). Changes in cytosolic (s), nuclear (n), mitochondrial (m) PKC (δ, ε) levels, and changes in total Akt and mGSK-3β phosphorylation after I/R were assessed by Western blot analysis. Senescence increased infarct size 50% in ovary-intact females (P < 0.05), whereas no differences in LV functional recovery or estradiol levels were observed. Ovx reduced functional recovery to a greater extent in aged compared with adult rats (P < 0.05). In aged (vs. adult), levels of m- and nPKC(-δ, -ε) were markedly decreased, whereas mGSK3β levels were increased (P < 0.05). Ovx led to greater levels of sPKC(-δ, -ε) independent of age (P < 0.05). I/R reduced p-Akt(Ser⁴⁷³) levels by 57% and increased mGSK-3β accumulation 1.77-fold (P < 0.05) in aged, ovary-intact females. These data suggest, for the first time, that estrogen alone cannot protect the aged female myocardium from I/R damage and that age- and estrogen-dependent alterations in PKC, Akt, and GSK-3β signaling may contribute to loss of ischemic tolerance.