Previous studies have shown that aqueous humor of calf and human eyes contains about 25 microM hydrogen peroxide. We have studied the removal of hydrogen peroxide by the outflow pathway of intact, freshly enucleated, calf eyes. Eyes were immersed under silicone oil that had a density greater than water and medium containing various agents was perfused into the anterior chamber. Medium passing through the trabecular meshwork and out the cut ends of the aqueous veins was trapped by the silicone oil and harvested. By measuring the concentration of hydrogen peroxide in the anterior chamber and in the emerging medium, we were able to study the rate of removal by the outflow structures and the effect of inhibitors on this rate. At 1 mM hydrogen peroxide, the amount emerging was undetectable by our methods. At 10 mM, the results were inconsistent, suggesting that tissue damage may have been occurring. At 5 mM, the concentration in the emerging medium was reduced 150-1000-fold, depending on time and conditions. This rate of removal could be reduced by 3-aminotriazole, reaching a maximum inhibition of about 50% at 80 mM. Addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) to further inhibit removal did not yield reliable results unless the concentration of H2O2 was lowered below 5 mM. Using loss of lactate dehydrogenase activity as a measure of cell damage, we found a 30% drop in activity after perfusing with BCNU, diamide, and 3-aminotriazole, followed by 3 hr with 10 mM hydrogen peroxide.(ABSTRACT TRUNCATED AT 250 WORDS)