ABSTRACTWe determined the binding affinities of multiple doses of four ophthalmic beta-blockers, timolol, betaxolol, levobunolol and carteolol, to the beta-1 and beta-2 receptors. With a Ki value of 0.39 nM and 0.36 nM for the beta-1 and beta-2 receptors, respectively, levobunolol shows the highest binding affinity to both beta receptors. The Kj values of timolol (1.97 nM for the beta-1 receptor and 2.00 nM for the beta-2 receptor) and of carteolol (0.83 nM and 0.85 nM for the beta-1 and beta-2 receptors, respectively) are characteristic of a nonspecific beta-blocker. On the contrary, betaxolol is a beta-1 specific antagonist (Kiof 23.33 nM) and has a very low binding affinity to the beta-2 receptor (Kiof 200.00 nM). With a radioreceptor assay, levels of beta-antagonist were measured in the plasma and aqueous humor 1 hour and 12 hours after instillation of 50 ul of 0.5% or 2% each of the four beta-blockers into the rabbit eye. At 1 hour after administration, the plasma levels of timolol, levobunolol, and carteolol are 9.89 ng/ml, 1.60 ng/ml and 8.00 ng/ml, respectively; such levels of 11.82 to 29.22 times the respective Ki values cause a virtually total blockade of both beta-1 and beta-2 receptors and suggest significant systemic absorption. Betaxolol has a peak 1 hour plasma level of 22.28 ng/ml, which is equivalent to only 3.08 times its Ki for the beta-1 receptor and 0.36 times its Kifor the beta-2 receptor; it has less systemic beta-1 blocking activity than the other three drugs and very minimal systemic beta-2 blocking activity. Peak aqueous humor concentrations of all 4 beta-blockers are extremely elevated at 1 hour after administration (timolol 1613.58 ng/ml; betaxolol 866.06 ng/ml; levobunolol 750.89 ng/ml; and carteolol 859.18 ng/ml). Such levels of 14 to 7192 times the respective Kivalues should cause a virtually complete blockade of both beta-1 and beta-2 receptors in the iris-ciliary body complex. At 12 hours after administration, plasma levels of all four beta blockers remain moderately elevated (timolol 0.94 ng/ml; betaxolol 9.43 ng/ml; levobunolol 0.66 ng/ml; and carteolol 1.61 ng/ml). Trough aqueous humor levels of levobunolol (43.38 ng/ml) and carteolol (92.81 ng/ml) remain elevated at least 300 times their Kivalue. On the contrary, 12-hour trough aqueous humor levels of timolol (33.67 ng/ml) and betaxolol (94.86 ng/ml) have decreased to 2 to 40 times their Kivalue, which may explain their requirement for twice daily administration clinically.