Endothelin, which has potent vasoconstrictor and mitogenic actions, was measured by radioimmunoassay in tissue extracts of sheep endometrium and myometrium and was found to be present in similar amounts in both tissues during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy 250–630 pg g⁻¹ wet weight). Immunoreactive endothelin extracted from endometrium eluted at the same position as standard endothelin-1 on reverse-phase HPLC. Immunohistochemical techniques demonstrated that during the oestrous cycle endothelin immunoreactivity was very low in caruncular and intercaruncular stroma, luminal epithelium, outer and inner glandular epithelium, myometrium and blood vessels until after day 12 (oestrus: day 0). Staining increased in all but the inner glands to day 16 and the most intense staining was found in intercaruncular luminal epithelium and outer glands and in myometrium, although endothelin in tissue extracts did not change over this period. During early pregnancy (days 4–20), staining in intercaruncular areas and in myometrium increased slightly from day 4 to day 12 to a maximum which was maintained from day 15 to day 20. Intensity of staining in caruncles increased only from day 15, particularly in the epithelium. Immunoreactive endothelin was also present in the trophoblast cells of the embryo on day 20 of pregnancy. Strong endothelin immunostaining was observed in uteri from ovariectomized ewes, particularly in epithelial cells and in blood vessels. The intensity of immunostaining in epithelium and stroma was increased slightly by oestradiol and decreased slightly by progesterone treatment, whereas treatment with oestradiol plus progesterone reduced staining intensity of endothelin in all types of tissue. Results of this study therefore demonstrate that immunoreactive endothelin-1 is present in the ovine uterus during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy and is localized in most types of cell. Endothelin-1 is regulated by ovarian steroids and pregnancy-related factors and may play an important role in the regulation of uterine blood flow, in uterine development and the establishment of pregnancy.