SUMMARY: The hypothesis that cyclic AMP plays an essential role in mediating the biological action of oestradiol on the uterus, was tested by determining the tissue concentration of the cyclic nucleotide after incubation of uteri of immature rats with oestradiol or after injection of this steroid into immature or ovariectomized rats. The effect of known stimulants of uterine adenyl cyclase, namely β-adrenergic drugs and prostaglandin E2 (PGE2), on the level of cyclic AMP in the uterus was also examined both in vitro and in vivo. In either system, oestradiol failed to enhance the concentration of cyclic AMP in the uterine tissue, whereas adrenaline or the almost purely β-adrenergic agonist isoprenaline (isoproterenol) caused cyclic AMP accumulation that was susceptible to inhibition by the β-adrenergic blocking agent propranolol. Prostaglandin E2, and to a much lesser degree prostaglandin F2α, increased cyclic AMP concentration in the uterus, but the effect of PGE2 was not inhibited by propranolol. It may be concluded that oestradiol does not cause appreciable stimulation of PGE2 synthesis or activation of β-adrenergic receptors in the rat uterus since, otherwise, increased cyclic AMP production should have been observed after the treatment with oestradiol. Isoprenaline mimicked the stimulatory action of oestradiol on uterine ornithine decarboxylase. However, this action of isoprenaline was abolished by propranolol, whereas that of oestradiol was only slightly, though significantly, inhibited. The present findings do not support the view that the action of oestradiol on the uterus is mediated by cyclic AMP, and also suggest that β-adrenergic receptors and PGE2 can have only a minor role, if any, in the mechanism of action of this hormone.