Dispersed human endometrial and myometrial cells grown for up to 4 months as monolayer cultures responded to addition of oestradiol-17β (1 × 10−9 mol/l) to the medium by increased [3H]thymidine incorporation into DNA, and an increased rate of cell division. Cultures derived from uterine leiomyomata showed a less consistent mitotic response to the hormone. Normal endometrial and myometrial cells grown in the oestradiolenriched medium showed a significantly higher efficacy of colony formation (increases of about 60 and 90%, respectively) than cells grown in control medium throughout the experimental period. Exposure to the hormone for 3–4 days was sufficient to induce this effect, which suggests that it does not depend on selection of hormone-sensitive cells. The mitogenic effect of oestradiol, as expressed by increased cloning efficacy, persisted for several days after transfer of the cells to a hormone-free medium. Cultures of foetal rat skeletal muscle failed to respond to oestradiol-17β in the cloning test, and oestradiol-17α was without effect on uterine cells. It is concluded that oestradiol-17β exerts a direct mitogenic action on some cellular components of the human endometrium and myometrium in vitro.