Beneficial effects of oestrogen administration on cognition are attenuated if treatment is initiated following long‐term ovarian hormone deprivation. The mechanisms underlying this attenuation are unknown. The present study aimed to assess the effects of long‐term ovarian hormone deprivation on the ability of subsequent oestradiol treatment to regulate oestrogen receptor (ER) α and ERβ, and steroid receptor coactivator (SRC)‐1 in the hippocampus and prefrontal cortex of middle‐aged rats. In an initial experiment to assess oestradiol regulation of these proteins, 2‐month‐old rats were ovariectomised and immediately implanted with capsules containing cholesterol or oestradiol. Brains were collected 10 days later. In a second experiment, middle‐aged (10‐month‐old) rats were ovariectomised or underwent sham surgeries. Five months later, sham‐operated rats were ovariectomised and received oestradiol implants. Previously ovariectomised rats underwent sham surgeries and received oestradiol or cholesterol implants. Protein levels of ERα, ERβ, and SRC‐1 were measured following 10 days of oestradiol treatment using western blotting. In young animals, oestradiol treatment significantly increased ERα in the hippocampus and prefrontal cortex relative to control treatment. In middle‐aged animals, immediate oestradiol treatment significantly increased ERα in hippocampus, but not the prefrontal cortex. However, delayed oestradiol treatment failed to significantly increase ERα protein levels in hippocampus, but did so in prefrontal cortex. Levels of ERβ and SRC‐1 were unaffected by oestradiol treatment in either brain area in either of the age groups. These data indicate that prolonged ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate ERα protein levels in brain areas important for cognition.