Isoprostanes (IsoPs) are isomers of prostaglandins that are generated from the free radical-initiated peroxidation of arachidonic acid (C20.4 ω-6). IsoPs exert potent bioactivity and are regarded as the "gold standard" to assess oxidative stress in various human diseases. Analogously, autoxidation of docosahexaenoic acid (DHA, C22.6 ω-3) generates an array of IsoP-like compounds that are termed neuroprostanes (NPs). A major class of NPs identified in vitro and in vivo contains F-type prostane rings and are know as F 4-NPs. A number of different F4-NP regioisomers are formed from the peroxidation of DHA. Among the eight possible regioisomeric groups, we hypothesize that 4- and 20-series NPs are generated in greater amounts than other classes because the precursors that lead to regioisomers other than those of the 4- and 20-series can be further oxidized to form novel dioxolane-IsoP-like compounds, analogous to those generated from arachidonate. Various mass spectrometric approaches, including electron capture atmospheric pressure chemical ionization mass spectrometry, were utilized to analyze NPs formed in vitro and in vivo based on their characteristic fragmentation in the gas phase. Experimental results were consistent with our hypothesis that 4- and 20-series NP regioisomers are preferentially generated. The discovery of regioselectivity in the formation of NPs will allow studies of the biological activities of NPs to focus on the more abundantly generated compounds to determine their role in modulating the pathophysiological consequences of DHA oxidation and oxidant stress.