Adenyl cyclase activity of rat pineal gland homogenates was measured from the rate of conversion of ATP-14C to radiolabeled cyclic 3',5'-AMP. Norepinephrine stimulated adenyl cyclase activity of male rat pineal glands to a greater extent than that of female rat pineals. Orchiectomy or testosterone treatment of either male or female rats failed to alter pineal adenyl cyclase activity, but ovariectomy slightly increased the stimulatory effect of norepinephrine. Administering estradiol to ovariectomized rats for 2 days inhibited the norepinephrine- and sodium fluoride-induced activation of adenyl cyclase. No inhibition was observed when the steroid was given 1 hr before sacrifice or when estradiol was added in vitro. Chronically administering progesterone had no effect on adenyl cyclase activity. Hypophysectomy failed to antagonize the stimulatory effect of ovariectomy and also failed to alter the inhibitory effects of estrogen treatment, indicating that estrogens do not act via release of gonadotropins. Norepinephrine activated pineal adenyl cyclase of rats in all stages of their estrous cycle except in proestrus, when estrogen levels are highest. This suggests that the physiological secretion of estradiol influences the norepinephrine-induced stimulation of pineal adenyl cyclase activity. (Endocrinology87: 527, 1970)