β-estradiol was administered to mice continuously by diffusion from a silastic tube that was implanted subcutaneously at 4 weeks of age. Four to 6 weeks of estrogen administration caused a substantial reduction in natural killer cell activity in the spleens from mice of either sex. Androgen (5α-dihydrotestosterone) did not. Castration of male or female mice did not affect natural killing and did not alter the effect of β-estradiol. Estradiol did not affect natural killing in vitro and the loss of natural killing was not due to a soluble or a cellular suppressor of natural killing. The effects of estradiol were not dependent on the thymus, since estradiol reduced natural killing in mice that had been neonatally thymectomized. After removal of the estrogen implant, natural killing recovered over a period of 8 weeks. The loss of natural killing may reflect a loss of bone marrow secondary to estrogen-induced osteosclerosis.