Increased production of oxygen free radicals may play a role in many diseases such as hypertension. As evidence indicates that xanthine oxidase may be involved in creating these reactive oxygen species, experiments were performed to additionally characterize hydrogen peroxide (H2O2) production in xanthine oxidase catalyzed reactions. In vitro measurements of hydrogen peroxide production from the xanthine/xanthine oxidase reaction were performed in buffered saline using an electrochemical technique, and the effect of allopurinol on inhibition of xanthine oxidase was determined. Experiments were also performed in blood plasma to characterize endogenous hydrogen peroxide producing capability and xanthine oxidase activity. In the presence of sodium azide, an inhibitor of catalase, peroxide production was measured in plasma after adding xanthine or xanthine oxidase and the results were similar to those obtained in buffered saline. When only sodium azide was added to plasma, hydrogen peroxide was produced at a level of 36.1 +/- 7.6 microM (n = 5). From these measurements, endogenous xanthine oxidase activity was estimated to be 6.5 +/- 0.3 mU/ml (n = 5). These results suggests that sufficient substrate exists in plasma to produce micromolar levels of hydrogen peroxide and xanthine oxidase may catalyze these reactions.