Oxidative stress, characterized by an imbalance between increased exposure to free radicals and antioxidant defenses, is a prominent feature of many acute and chronic diseases and even the normal aging process. However, definitive evidence for this association has often been lacking due to recognized shortcomings with methods previously available to assess oxidant stress status in vivo in humans. Several in vitro markers of oxidative stress are available, but most are of limited value in vivo because thay lack sensitivity and/or specificity or require invasive methods. Isoprostanes (IsoPs) are prostaglandin (PG)-like compounds that are produced in vivo independently of cyclooxygenase enzymes, primarily by free radical-induced peroxidation of arachidonic acid. F2-IsoPs are a group of 64 compounds isomeric in structure to cyclooxygenase- derived PGF2α. Other products of the IsoP pathway are also formed in vivo by rearrangement of labile PGH2-like IsoP intermediates including E2- and D2-IsoPs, cyclopentenone-A2- and J2-IsoPs, and highly reactive acyclicketoaldehydes (isoketals). Oxidation of docosahexaenoic acid, an abundant unsaturated fatty acid in the central nervous system, results in the formation of IsoP-like compounds, termed neuroprostanes. Measurement of F2-IsoPs is the most reliable approach to assess oxidative stress status in vivo, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. Moreover, F2-IsoPs and other products of the IsoP pathway exert potent biological actions both via receptor-dependent and independent mechanisms and therefore may be pathophysiological mediators of disease. Measurement of F2-IsoPs may provide a uniquely valuable approach to understanding of the clinical pharmacology of antioxidants.