The ocular penetration of two beta-adrenergic blockers, befunolol and timolol, was studied using albino and pigmented rabbits. A 1% ophthalmic solution of 14C-befunolol hydrochloride or 14C-timolol maleate was instilled in one eye of nonsedated rabbits, and the concentrations of radioactive material in the ocular tissues were determined at various intervals. In the albino rabbit, the time courses of the concentration changes in the cornea and aqueous humor could be analyzed on the basis of a compartment theory, and the following coefficients were calculated using a computer; i.e., permeability of the corneal epithelium (kep), the distribution volume in the anterior chamber (Va), the cornea-aqueous transfer coefficient in reference to the corneal volume (kc.ca), the aqueous-cornea transfer coefficient in reference to Va (ka.ac), the loss rate from the anterior chamber (ko) and the steady state distribution ratio between the aqueous and cornea (rac). The following values were obtained for befunolol: kep--2.6 x 10(-3) cm hr-1, kc.ca--0.51 hr-1, ka.ac--0.18 hr-1, ko--2.7 hr-1, Va--250 microliters and rac--0.9. Those of timolol were as follows: kep--2.3 x 10(-3) cm hr-1, kc . ca--0.82 hr-1 and ko--2.5 hr-1. The estimates for Va and rac could not be calculated from the data obtained in the timolol experiment. Assuming that Va and rac for befunolol were also applicable for timolol, ka.ac for timolol was calculated to be 0.25 to 0.29 hr-1. The beta-adrenergic blockers tested here penetrated the cornea rather easily and they were lost from the anterior chamber mainly by diffusion, probably into the blood circulation through the anterior uvea. The data obtained in the pigmented rabbit indicated that these beta-adrenergic blockers are bound to the melanin-containing ocular tissues and are released from there very slowly. It is suggested that in heavily pigmented subjects, beta-adrenergic blockers might be less effective after short-term use and, furthermore, intraocular binding may occur after long-term use.