In 1990 we discovered the formation of prostaglandin F₂-like compounds, F₂-isoprostanes (F₂-IsoPs), in vivo by nonenzymatic free radical–induced peroxidation of arachidonic acid. F₂-IsoPs are initially formed esterified to phospholipids and then released in free form. There are several favorable attributes that make measurement of F₂-IsoPs attractive as a reliable indicator of oxidative stress in vivo: (i) F₂-IsoPs are specific products of lipid peroxidation; (ii) they are stable compounds; (iii) levels are present in detectable quantities in all normal biological fluids and tissues, allowing the definition of a normal range; (iv) their formation increases dramatically in vivo in a number of animal models of oxidant injury; (v) their formation is modulated by antioxidant status; and (vi) their levels are not effected by lipid content of the diet. Measurement of F₂-IsoPs in plasma can be utilized to assess total endogenous production of F₂-IsoPs whereas measurement of levels esterified in phospholipids can be used to determine the extent of lipid peroxidation in target sites of interest. Recently, we developed an assay for a urinary metabolite of F₂-IsoPs, which should provide a valuable noninvasive integrated approach to assess total endogenous production of F₂-IsoPs in large clinical studies.