New Zealand white rabbits have a circadian rhythm of intraocular pressure; pressure is higher during the dark than during the light. We explored the possiblity that the dark phase increase of serum and/or ocular melatonin plays a role in regulating the rhythm of intraocular pressure. Exogenous melatonin was delivered by four routes: topical application (0.1 and 10μg), intravenous injection (1μg), intravitreal injection (9μg) and intra-arterial infusion (50 ng and 1μg). Melatonin delivered by these routes did not increase intraocular pressure. We also measured the concentration of melatonin in the aqueous after unilateral intra-arterial infusion to confirm that melatonin delivered by this route reached the eye. The concentration was 419±99 and 109±17 pg/ml (n = 8, p<0.025) in the ipsilateral and contralateral eyes, respectively. The concentration of melatonin in the aqueous after intra-arterial infusion of saline was less than the sensitivity of the assay (<16 pg/ml, n = 6). The results of these experiments indicate that melatonin does not increase IOP significantly under the experimental conditions employed in this study.