The ocular and systemic disposition of levobunolol (LBUN), an antiglaucoma agent, was studied in albino rabbits. After topical administration to eyes, LBUN was rapidly adsorbed, with 2.5% of the dose bioavailable to the intraocular tissues as intact drug and 46% to the systemic circulation. On passage across the cornea, approximately 4.7% of a topically applied LBUN dose was biotransformed to dihydrolevobunolol (DHB), and subsequently became bioavailable to intraocular tissues. The major sites of ocular metabolism were the cornea epithelium and the iris-ciliary body. Another 12% of the topical LBUN dose entered the systemic circulation as DHB after presystemic biotransformation. Our study indicated a rapid absorption of LBUN into the aqueous humor after topical dosing. The tpeak was 15 min after dosing and the Cmax was 4 micrograms/mL. Dihydrolevobunolol (DHB) was formed steadily and reached a maximum in the aqueous humor 45 min after dosing. After distribution equilibrium had been reached, the aqueous humor concentrations of both LBUN and DHB declined. Six hours after dosing, the concentration of DHB in the aqueous humor was approximately 10 times higher than that of its parent compound. Because DHB is equivalent to its parent compound in beta-blocking activity, its formation in the rabbit eye may contribute to the pharmacodynamic effects observed after topical doses of LBUN.