For differential imaging of mammary tumors with estrogen receptors and without estrogen receptors we required γ-emitting estrogen analogues. In this paper we report on the binding properties of 7α-, 16α-, and 17α-methylselenoestrogens and 17α-phenylselenoestrogens relative to the binding properties of estradiol.The selenium-containing estrogens retained the ability to displace [3H]estradiol from the estrogen receptor of rabbit uterine cytosol, although in most instances the displacement was small (3–7% compared to estradiol).The most active compounds were 16α-phenylselenoestrone, 16α-methylselenoestradiol, and 17α-methylselenomethyl-estradiol which had relative binding of 23, 27, and 31%, respectively, compared with that of estradiol.16α-Methylselenoestradiol was able to translocate the estrogen cytosol receptor to the nucleus, in vitro, but was not able to increase the uterine weight when administered to mice in vivo.