The interaction of the steroid alkylating agent estradiol mustard (EM) with estrogen receptor was investigated under cell-free conditions. EM failed to bind to cytoplasmic estrogen receptor obtained from either rat uterus or human breast-cancer tissue. Nevertheless, in vivo studies in the rat uterus demonstrated that intraperitoneal injections of EM translocated estrogen receptor into the nucleus within 10 hours. Estradiol injections caused a similar depletion of cytosol receptor and transfer to the nucleus, but this translocation was completed within 2 hours. These studies suggest that, in vivo, EM is slowly converted to a metabolite, possibly free estradiol, which binds to estrogen receptor and transfers it to the nucleus.