The endogenous synthesis of peroxynitrite (ONOO(-)) has been implicated in a number of diseases, but assessments of its cytotoxicity and genotoxicity have been hampered by its extremely short half-life under physiological conditions (<20 ms) and the consequent difficulty in exposing cells to known concentrations of it over at least several hours. Two methods for peroxynitrite delivery to cell cultures were investigated, one involving steady infusion of preformed ONOO(-) and the other based on the continuous in situ synthesis of ONOO(-) from NO and O(2)(-). In the latter, NO was supplied by diffusion through gas permeable tubing and O(2)(-) was generated using the hypoxanthine-xanthine oxidase reaction. The performance of both methods was assessed by measuring the rates of formation of tyrosine derivatives (dityrosine and nitrotyrosine) that are commonly employed as biomarkers for peroxynitrite. Experimental results in the absence of cells were compared in each case with predictions from kinetic models. In the infusion system, the measured dityrosine and nitrotyrosine yields were in excellent agreement with those predicted from the model. To characterize the other system, experiments were performed first to determine the kinetics of hypoxanthine oxidation by xanthine oxidase and uric acid oxidation by uricase. Simulations of the complex reaction network in the complete synthesis system suggested that dityrosine should be the major product there, that the yields of both tyrosine derivatives should be very sensitive to the relative rates of NO and O(2)(-) delivery, and that equal rates for NO and O(2)(-) should maximize those yields. Experiments performed under the predicted optimal conditions showed much lower levels of dityrosine than expected and no detectable nitrotyrosine. The unexpectedly low yields of tyrosine products could be explained largely by the partial inactivation of both xanthine oxidase and uricase by peroxynitrite-derived NO(2) and CO(3)(-) radicals. We conclude that continuous infusion of peroxynitrite is the more promising approach.