Estrogen deficiency during menopause is often associated with memory dysfunction. However, inconsistencies regarding the ability of estrogen to improve memory in menopausal women highlight the need to evaluate, in a controlled animal model, the potential for estrogen to alleviate age-related mnemonic decline. The current study tested whether estrogen could ameliorate spatial reference memory decline in aged female mice. At the conclusion of testing, levels of the presynaptic protein synaptophysin, and activities of the synthetic enzymes for acetylcholine and GABA, were measured in the hippocampus and neocortex. Aged (27-28-month-old) female C57BL/6 mice were given daily subcutaneous injections of 1 microg or 5 microg of beta-estradiol-3-benzoate dissolved in sesame oil. Control mice received daily injections of sesame oil or no injections. Estradiol treatment began 5 days prior to behavioral testing and continued throughout testing. Spatial and non-spatial memory were assessed in the Morris water maze. The 5 microg dose of estradiol significantly improved spatial learning and memory in aged females. The performance of 5 microg females improved significantly more rapidly than that of control females; estradiol-treated females performed at asymptotic levels by session 2. Furthermore, 5 microg females exhibited a more robust spatial bias than controls during probe trials. In contrast, 1 microg of estradiol did not improve spatial task performance. Neither dose affected performance of the non-spatial task. In the hippocampus, synaptophysin was increased in 5 microg females relative to controls. Estrogen did not affect enzyme activities in either brain region. This study is the first to examine the effects of estrogen replacement on spatial reference memory and synaptophysin expression in aged post-estropausal female rodents. The results suggest that: (1) estrogen can profoundly improve spatial reference memory in aged females, and (2) this improvement may be related to increased hippocampal synaptic plasticity, but not modulation of the synthetic enzymes for acetylcholine and GABA.