The effect of dexamethasone treatment on glycosaminoglycans (GAG) in the human trabecular meshwork was studied by placing 20 pairs of eyes in perfusion organ culture. One eye received 550 nM dexamethasone in addition to culture medium; the fellow eye received culture medium only. 3H-glucosamine and 35S-sulfate were added to the medium for the final 48 hr of culture. The meshwork was then dissected, and the GAGs were isolated and subjected to sequential enzymatic degradation. Active labeling of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate was found in both control and steroid-treated eyes. Dexamethasone-treated eyes had an average 92% increase in the 3H-glucosamine incorporation rate in the undigestible GAG residue fraction after 14-21 days treatment (20.6% versus 10.7%, P = 0.03). This change was not apparent in eyes treated for 7 days. In this preliminary study, dexamethasone appeared to cause a time-dependent increase in the undigestible GAG incorporation profile in human trabecular meshwork.