Objective To explore the impact of combination use of prostaglandin analogue and cholinergic agonists on main matrix metalloproteinases (MMPs) synthesized by albino rabbit ciliary muscle. Methods Normal adult albino rabbits were divided into the control group, 2%pilocarpine group, 0.004% travoprost group and travoprost plus pilocarpine group. Two rabbits in the control group were executed after treated with normal saline for one day. Two rabbits were separately executed on the 7th, 14th and 24th day of the treatment in each drug treated group. In each subgroup ciliary muscle band of 4 eyes was taken and made into homogenate. The MMPs activities of 10 subgroups were assayed by zymography. Bands’ intensity which represents the activity of MMPs was measured by the UltraViolet Illumination system. Results A bright band of MMP-1/2 was showed on each lane at the position corresponding to the molecular weight of 62 kD in the ciliary smooth muscles electrophoresis. When ion Zn and Ca was displaced by MMPs inhibitor EDTA, this bright band disappeared. Compared with the control group, MMP1/2 activity increased by 4.0%, 4.1%and 14.0%after 7, 14 and 24 days of pilocarpine treatment. Corresponding data was 23.2%, 61.7% and 111.5% in the travoprost group and 49.3%, 68.0% and 88.4% in the travoprost plus pilocarpine group. Conclusions Pilocarpine has little effect on activity of MMP1/2. Travoprost can increase activity of MMP1/2 gradually. Activity of MMP1/2 is rapidly increased by pilocarpine combined with travoprost, but shows small change with the prolonged treatment.