The influence of treatment with oestradiol on the effects of the uterine relaxants, relaxin, salbutamol (an agonist at β2-adrenoceptors) and cromakalim (a potassium channel opener) and their interactions with the uterine stimulant oxytocin were investigated in vivo in the ovariectomized rat. Oestradiol benzoate (0·4 μg/kg per day) significantly increased sensitivity to cromakalim as an inhibitor of spontaneous uterine contractions compared with vehicle-treated rats by approximately threefold. The same dose of oestradiol benzoate had no effect on uterine sensitivity to salbutamol. Previous studies have shown that this dose of oestradiol benzoate produces a twofold increase in uterine sensitivity to relaxin as an inhibitor of spontaneous contractions. Oestradiol influenced the ability of relaxin to inhibit oxytocin-stimulated uterine contractions. In corn oil-treated rats, uterine responses to relaxin were markedly reduced during oxytocin infusion compared with responses to relaxin before oxytocin; the maximum obtainable response to relaxin was less than 50% inhibition. In oestradiol-treated rats, uterine sensitivity to relaxin during oxytocin infusion was similar to that observed against spontaneous contractions. Cromakalim was able to inhibit uterine contractions during oxytocin infusion in both corn oil- and oestradiol-treated rats, uterine sensitivity to cromakalim being similar in the absence and presence of oxytocin for both hormone treatment groups. Salbutamol was also able to inhibit uterine contractions during oxytocin infusion in both corn oil- and oestradiol-treated rats. Oestradiol treatment increased the potency of salbutamol as an inhibitor of oxytocin-stimulated uterine contractions compared with corn oil treatment by 3·5-fold. The interaction of oestradiol and relaxin during late pregnancy may be important for attenuation of the myometrial response to stimulants.