The formation of new metabolites of the stable nitroxyl radical 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) inside the isolated perfused rat liver was examined. The paramagnetic 4-hydroxy derivative (TEMPOL) and the diamagnetic 1,4-dihydroxy derivative were found to be the major metabolites besides the well-known corresponding hydroxylamine of TEMPONE. No reoxidation of the hydroxyl group in the 4-position was observed. The conversion of nitroxides to the sterically hindered secondary amines remains speculative. A redox cycle of nitroxide and hydroxylamine including the secondary amines is discussed. For the first time the biotransformation of the stable nitroxyl radical TEMPONE detected by means of GC and GC-MS has been examined and new metabolites have been described, i.e. the newly discussed metabolites have to be considered for the interpretation of electron paramagnetic resonance (EPR), magnetic resonance imaging (MRI) and dynamic nuclear polarization (DNP) measurements on using the spin probe TEMPONE.