The rate of aqueous humor formation was determined in the cynomolgus monkey eyes by a tracer dilution technique. One 25-gauge needle was inserted into the posterior chamber and a solution of fluorescein labeled dextran with a molecular weight of 40,000 was infused at a constant rate. The aqueous humor was collected through a needle inserted into the anterior chamber, while the intraocular pressure (IOP) was maintained at a constant level. The aqueous humor formation rate and the dye distribution volume were calculated from the time profile of the dye concentration in the effluent aqueous humor. By means of this technique, the effects of pilocarpine, salbutamol, and timolol on the aqueous humor formation rate were studied. The test drug solution was administered into the conjunctival reservoir during a 90-min period before measurements and also during the measurement so that a steady-state drug concentration was maintained in the anterior chamber during the measurement. Pilocarpine 0.1% reduced the aqueous humor formation rate to approximately 50% of the control without significantly changing the IOP or the distribution volume. Salbutamol 0.5%, a beta-adrenergic agonist, increased the rate by about 38%, but timolol 0.1%, a beta-adrenergic antagonist, reduced the rate by an average of 36%. Timolol caused a statistically significant lowering of the IOP by about 2 mmHg. Simultaneous administration of salbutamol 0.5% and timolol 0.2% caused no change in the aqueous humor formation rate or the IOP. The barrier function of the blood aqueous barrier was not altered by these drugs as revealed by aqueous protein determinations.