V-C horizontal diffusion cell has been used to carry out the initial study of timolol across intact and stripped hairless mouse skin and human cadaver skin. Also, we have systematically studied the effect of some factors such as hydration time, medium pH value as well as various transdermal enhancers on the permeability of timolol in vitro. We investigated the determination method of timolol by RP-HPLC using YWG-C18 column with water-acetonitrile-triethylamine (87:13:1) as the mobile phase. The results indicated the feasibility of transdermal absorption of timolol. The permeabilities of timolol raised significantly with properly extending of hydration time, increasing of medium pH and with presence of various kinds of transdermal enhancers. With 10% propylene glycol and 1% azone were used conjunctly, they showed obviously synergistic effects. All these studies contributed theoretically and practically to the further development and utilization of timolol in the form of transdermal patch.