The effects of timolol on the aqueous humor (AH) formation and AH outflow in cat eyes were simultaneously studied with a continuous infusion method under constant intraocular pressure. The rate of AH formation was reduced 28%, 56%, and 71%, respectively, by 0.005%, 0.025%, and 0.15% of timolol solution infused intracamerally. There was no significant change of AH outflow after the administration of timolol. It was noted that the pupil was dilated by 0.025% and 0.15% timolol solutions. To determine the action mechanism of timolol to inhibit AH formation, experiments of carbonate dehydratase inhibition were performed with various concentrations of timolol. No appreciable enzyme inhibition was noted with timolol at concentrations up to 0.5%. It is thus concluded that the action mechanism of timolol to inhibit AH formation differs from that of carbonate dehydratase inhibitors.