Two timolol preparations, a gel and an eyedrop with a thickening agent, and one commercial eyedrop without a thickening agent, were studied in rabbits. After topical administration of these three preparations in rabbits, aqueous humor was withdrawn and the proteins removed from the samples by precipitation with acetonitrile. Timolol concentrations were determined directly by an HPLC method. The HPLC mobile phase was composed of methanol and 5 mM d-camphorsulfonic acid (in 1% acetic acid) with a ratio of 49:51 (v/v). A reversed phase C18 column was used to separate samples with a flow rate of 0.8 mL/min and a UV detector set at 284 nm. A two-compartment pharmacokinetic model was used to fit the aqueous humor level for determining the drainage (kd) and absorption rate constants (ka) in the precorneal area as well as the elimination rate constant (ke) of timolol in aqueous humor. For ka +kd, the eyedrop without a thickening agent had the highest value (0.160 min-1), followed by the eyedrop with a thickening agent (0.030 min-1), and the gel had the lowest value (0.009 min-1). It suggests that the gel has a longer retention time in eyes to improve ocular bioavailability and decrease side effects. The AUC0 approximately infinity for the aqueous humor profile with time coordinates were 4142, 2974, and 1604 micrograms min/mL, for the gel, the eyedrop with a thickening agent, and the eyedrop without a thickening agent, respectively. In another study, timolol preparations were also topically administered in alpha-chymotrypsin-induced glaucoma rabbits for determining the lowering effect on intraocular pressure (IOP). The durations of depressing IOP for the gel, the eyedrop with a thickening agent, and the eyedrop without a thickening agent were 24, 14 and 10 hrs, respectively. Thus, the gel preparation has a longer duration and a higher ocular bioavailability which might be further developed in the treatment of open-angle glaucoma.