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We evaluated the effects of three specific glycosaminoglycan-degrading enzymes, chondroitinase ABC, testicular hyaluronidase, and heparinase, on the intraocular pressure (IOP) and the anterior chamber structures of bovine eyes. The anterior segment of freshly enucleated bovine eyes was perfused in a modified organ culture system. After each enzyme solution or its corresponding vehicle was introduced into the anterior chamber, the IOP was monitored for ≤6 h. Compared to the control group, the chondroitinase ABC-treated group showed a significant IOP reduction after 1 h of perfusion. Testicular hyaluronidase and heparinase did not show any significant effect. Light microscopy revealed that chondroitinase ABC and testicular hyaluronidase induced detachment of the endothelial cells from trabecular beams in the uveal meshwork. Disruption of the tissues in the juxtacanalicular region and fragmentation of the endothelial cells facing the angular aqueous plexus were observed in the chondroitinase ABC- and heparinase-treated specimens. Electron microscopic study revealed detachment of trabecular meshwork cells from the beams and rupture of the inner wall of the aqueous plexus in all the enzyme-treated eyes. Cellular organelles and architectures were better preserved in chondroitinase ABC-treated eyes than those treated with hyaluronidase and heparinase. These results indicate that chondroitinase ABC has a pronounced effect on the IOP with relatively minor effect on the trabecular meshwork structures.