Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase–glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/˙OOH) to the corresponding hydroxyl radical (HO˙) adducts (nitrone/˙OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/˙OH] and [DIPPMPO/˙OH]. Additional nitrone/˙OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/˙OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/˙OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis:trans-nitrone/˙OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/˙OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/˙OOH. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO˙ formation in biological systems.