Topical application of sulprostone, a preferential prostaglandin EP3receptor agonist, caused a dose-dependent reduction of the circadian elevation of intraocular pressure (IOP) in New Zealand albino rabbits which were entrained to 12-hr/12-hr light-dark environment. Corresponding to the effect on IOP, 0.2μg, 2μg, and 20μg sulprostone decreased the norepinephrine (NE) concentration in the aqueous humor in the dark phase. There was no breakdown of the blood-aqueous barrier. Bilateral applications of 2μg and 20μg sulprostone to entrained rabbits that had undergone unilateral, preganglionic transection of the cervical sympathetic trunk reduced the circadian IOP elevation in the intact eye, but caused little IOP change in the decentralized eye. These results indicate that the lOP-lowering effect of topical sulprostone in rabbits is dependent on sympathetic neural activity and that prejunctional inhibition of NE release may be an important mechanism of action.