Horse cytochrome c was reacted with the spin label (succinimidyl-2,2, 5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys8, Lys25, Lys72, Lys86 or Lys87, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys8 and Lys87 only one component contributes to the spectrum whereas for Lys25, Lys72 and Lys86 the spectrum consists of two components. The highest mobility with the rotational correlation time, tauB, of 0.38 ns was observed for Lys87. The longest tauB of 1.84 ns was obtained for Lys72. An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.