Abstract: A matrix-type transdermal therapeutic system of timolol (TTS-timolol) was well prepared. The patch consisted of backing membrane layer, timolol reservoir layer, pressure sensitive adhesive layer and protective layer. A sensitive and reliable HPLC-UV method for the determination of plasma level of timolol in healthy volunteers was developed. Effective therapeutic plasma level of timolol (4 ng/ml) was attained 4 h after application of the timolol patches and was maintained within 32 h while the patch was removed at 24 h. The pharmacokinetic behavior of this transdermal therapeutic system (TTS)-timolol in human showed zero order absorption and well fitted to a one compartment model. The pharmacokinetic parameters are: T_max= 18. 8h; C_max= 11.2 ng/ml; AUC= 265. 7 ng/ml·h; V_ss=120. 0L; K=0. 084 h_-1. In comparison with the results of oral administration of timolol tablets, TTS-timolol possesses some advantages: stable plasma level, long effective time and convenient administration.