SUMMARY: High-affinity binding of [2,4,6,7-3H]oestradiol-17β has been studied in cytosols prepared from hypothalami, pituitaries and uteri of female rats killed at different stages of the oestrous cycle, and in cytosols prepared from the hypothalami and pituitaries of male rats. In all cases the equilibrium dissociation constant of reaction was of the order of 10−10 mol/l. The number of available high affinity sites per tissue (n) varied with physiological state. In females, n fluctuated with the oestrous cycle. In hypothalamus and pituitary, n fell by about 60 and 40% respectively in pro-oestrus, replenishment occurred during oestrus but could be delayed by phenobarbitone administration during the afternoon of pro-oestrus. In the uterus, n varied biphasically, there being peaks during dioestrus and oestrus, and troughs at pro-oestrus and metoestrus. The numbers of available sites at metoestrus were 12·5 × 109, 10·6 × 1010 and 24·4 × 1010 for hypothalamus, pituitary and uterus respectively. In male rats, values for n were similar to those obtained for females at pro-oestrus (n/hypothalamus = 6·8 × 109, n/pituitary = 4·2 × 1010). Binding was oestrogen specific in all the tissues studied.