The aqueous humor formation rate was determined in the anesthetized rabbit with a tracer dilution technique. The anterior chamber was cannulated with two 25-gauge needles: one was inserted into the posterior chamber through the pupil and the other was placed in the anterior chamber near the iridocorneal angle. Fluorescein-dextran solution was infused into the posterior chamber at a constant rate and the aqueous humor was collected through the anterior chamber needle. The aqueous humor formation rate and the volume of dye distribution were calculated simultaneously from the dye dilution curve. Utilizing this technique, the effects of the following substances on aqueous humor formation were studied following topical administration to the conjunctival reservoir: cholinomimetic drugs, pilocarpine and physostigmine; the parasympatholytic drug, atropine; adrenoceptor agonists, norepinephrine, epinephrine, isoproterenol and salbutamol; and beta-adrenoceptor antagonists, timolol and propranolol. Cholinomimetic drugs, i.e., pilocarpine 0.1 mg ml-1 to 10 mg ml-1 and physostigmine 1 mg ml-1, increased the aqueous humor formation rate, while atropine 1 mg ml-1 decreased the rate. The increase in aqueous humor formation by the cholinomimetics was completely antagonized with simultaneous administration of atropine. Norepinephrine 5 mg ml-1 and epinephrine 5 mg ml-1 reduced significantly both the aqueous humor formation rate and intraocular pressure (IOP). Isoproterenol 5 mg ml-1 caused a significant lowering of the blood pressure and IOP and tended to lower aqueous humor formation, although the decrease was not statistically significant. No significant effect on the blood pressure, IOP or aqueous humor formation was observed with isoproterenol 2 mg ml-1. Salbutamol 5 mg ml-1 raised the IOP while timolol 1 mg ml-1 lowered the IOP without any significant change in aqueous humor formation or blood pressure. Timolol 0.5 mg ml-1 or propranolol 5 mg ml-1 did not cause a significant effect on the blood pressure, IOP or aqueous humor formation rate.