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We compared the concentration of hyaluronic acid in the aqueous humor of primary open-angle glaucoma (POAG) patients and non-glaucomatous patients. Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled POAG (POAG group). Aqueous humor (0·1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber. The same procedure was performed for 22 non-glaucomatous patients just before cataract surgery (control group). Immediately after collection, the aqueous humor was stored at −20°C. The concentration of hyaluronic acid was determined by a sensitive, noncompetitive and nonisotopic fluoroassay. The median (range) concentrations of hyaluronic acid of the POAG and control groups were 298·4 μg L−1 (99·0–743·7 μg L−1) and 545·1 μg L−1 (145·0–2366·0 μg L−1), respectively. The difference in concentrations of hyaluronic acid between the groups was statistically significant (P<0·001). In conclusion, the concentration of hyaluronic acid in the aqueous humor in POAG patients is lower than in non-glaucomatous patients. Further studies are necessary to determine the role of hyaluronic acid in the pathophysiology of POAG.