The goal of the present study was to explore the effects of long-term hormone deprivation on the ability of subsequent estrogen replacement to affect cognition. Female rats, 12 months of age, underwent ovariectomies (n = 30) or sham surgeries (n = 10). Intact rats and 20 ovariectomized rats received cholesterol implants. Ten ovariectomized rats received implants containing 25% estradiol. Five months later, implants were replaced. Half of the ovariectomized rats with cholesterol implants received estradiol implants and half received new cholesterol implants. Rats with estradiol implants received new estradiol implants. Intact rats were ovariectomized and given estradiol implants. Beginning 1 wk later, working memory performance was assessed in an eight-arm radial maze across 24 d of acquisition and during eight additional trials in which a 2.5-h delay was imposed between the fourth and fifth arm choices. Estradiol replacement initiated immediately after ovariectomy at either 12 or 17 months of age significantly improved performance during acquisition and delay trials, compared with control treatment. When estradiol replacement was initiated at 17 months of age, 5 months after ovariectomy, no enhancements were evident. Uteri of rats that experienced delayed estradiol replacement weighed significantly more than uteri of ovariectomized controls but significantly less than uteri of rats that received immediate estradiol replacement. Uterine weight negatively correlated with mean errors during acquisition. These results indicate that whereas chronic estradiol replacement regimens positively affect working memory in middle-aged animals when initiated immediately after ovariectomy, estradiol replacement is not effective when initiated after long-term hormone deprivation.