Here we provide the first evidence, to our knowledge, that estradiol (E(2)) affects learning and memory via the newly discovered estrogen receptor beta (ERbeta). In this study, ERbeta knockout (ERbetaKO) and wild-type littermates were tested for spatial learning in the Morris water maze after ovariectomy, appropriate control treatment, or one of two physiological doses of E(2). Regardless of treatment, all wild-type females displayed significant learning. However, ERbetaKOs given the low dose of E(2) were delayed in learning acquisition, and ERbetaKOs administered the higher dose of E(2) failed to learn the task. These data show that ERbeta is required for optimal spatial learning and may have implications for hormone replacement therapy in women.