F₂-isoprostanes are complex metabolites of arachidonic acid generated via nonenzymatic free radical oxidation and are isomeric to prostaglandin F_2α, enzymatically produced by prostaglandin H₂ synthase. In theory, four distinct regioisomeric families are possible. These regioisomeric families have a common 1,3-diol cyclopentane structural feature, but differ by the comparative length of two attached alkyl chains and the position of a third hydroxyl group. Eight synthetic PGF_2α isomers were found separable by capillary gas chromatography (GC) and reversed-phase high-performance liquid chromatography (HPLC). Electrospray ionization tandem mass spectrometry was used to detect the elution of these isomers from the HPLC column by monitoring the characteristic loss of 44 u (C₂H₄O) from the 1,3-diol cyclopentane ring. Catalytic reduction, derivatization, and electron ionization mass spectrometric techniques were used to obtain definitive information as to the location of the side chain hydroxyl position in these isomers through abundant α-cleavage ions. Free radical oxidation of arachidonic acid was used to generate a complex mixture of F₂-isoprostanes, which were separated by HPLC and capillary GC. Members of each of the four specific regioisomeric isoprostane families could be identified in this mixture from the predicted α-cleavage ions. Although many epimers within a single family type could be separated, the four regioisomeric families were substantially superimposed in their HPLC and GC elution. The Type I and Type IV regioisomers were the major F₂-isoprostane products, but the complexity of the isomers required more than a simple GC-mass spectrometry assay to precisely identify a particular stereoisomer within a regioisomeric family (e.g., 8-epi-PGF_2α). Type I F₂-isoprostanes are unique noncyclooxygenase products and may be more specific targets to measure lipid peroxidation in vivo.