Estradiol-17β (E2) plays critical roles in female maturation, sexual receptivity, ovulation and fertility. In many mammals, contact with males can similarly affect these female parameters, whereas male excretions contain significant quantities of E2. We administered radiolabeled estradiol ([3H]E2) to male mice in doses representing a small fraction of their endogenous E2. These males were paired with sexually receptive females, and radioactivity was traced into the females’ systems. In Experiment 1, males were given [3H]E2 at 24 and 1 h before mating. Male-to-female [3H]E2 transfer intensified with increasing numbers of intromissions and spiked in the uterus after insemination. In Experiment 2, sexually experienced young males received [3H]E2 at 72 and 24 h before mating, and all mated to ejaculation. The copulatory plug deposited in the female reproductive tract contained substantial levels of radioactivity. The uteri, other tissues and blood serum of females displayed radioactivity indicative of E2 transfer. In Experiment 3, radioactivity was observed 3 and 18 h after insemination in the females’ uteri and other tissues, including parts of the brain. In Experiment 4, we observed substantial levels of radioactivity in semen as well as the copulatory plugs retrieved from the females after mating. Transferred E2 could directly affect abundant estrogen receptors in the female reproductive tract without potential metabolism by the liver. Sexually transferred E2 may facilitate uterine preparation for blastocyst implantation. These data converge with several lines of evidence indicating that male-sourced E2 can transfer to proximate females in bioactive form, contributing to various mammalian pheromonal effects.