The effect of timolol on the active transport system in the iris root-ciliary body of rabbits was studied to elucidate the action mechanism of timolol. Neither Na<sup>+</sup>-K<sup>+</sup>-adenosine triphosphatase (ATPase) nor Mg<sup>++</sup>-ATPase was inhibited by timolol at 1 × 10<sup>––4</sup> M concentration. None of the energy production parameters (oxygen consumption, glucose metabolism, and lactic acid formation) was inhibited by timolol either. Further, the biosynthesis of prostaglandins E<sub>2</sub> and F<sub>2α</sub> was not affected by timolol at 1 × 10<sup>––3</sup> M. The blood flow to the eye was measured with a <sup>85</sup>Sr-microsphere method. It was found that the blood flow in the iris root-ciliary body and choroid was significantly reduced by a topical application of 0.25% timolol. The dopamine concentration in the iris root-ciliary body was reduced by timolol at 1 × 10<sup>––5</sup> M concentration. Neither epinephrine nor norepinephrine concentration was altered by timolol. The results indicate that timolol reduces the rate of aqueous humor formation through reduction of blood flow to the ciliary process rather than via the inhibition of the active transport system or that of prostaglandin biosynthesis.