Estetrol (E4), a naturally occurring estrogen produced exclusively by human fetal liver, is currently being evaluated for potential use in contraception and menopausal care in humans. The present study was designed to profile E4 effects on the central nervous system, to assess the in vivo effects of E4 administration on Beta-Endorphin (β-END) release in specific brain structures and to evaluate whether E4 has synergic or antagonistic effects on estradiol-mediated β-END synthesis.