The retention behaviour of equine, tuna, canine and bovine cytochrome c and the corresponding equine and tuna apocytochrome c has been investigated using reversed-phase gradient elution chromatographic procedures. A range of operating temperatures between 5 and 85°C were utilised to monitor the influence of protein unfolding on the corresponding retention parameters. Chromatographic measurements were obtained with both an n-octadecyl (C8) and n-butyl (C4) sorbent in order to investigate the role of ligand structure on protein retention. The results demonstrated significant differences in the S and log k0 values between all cytochrome c proteins despite the very high degree of sequence homology. These observations suggest that specific amino acid substitutions are directly involved in the chromatographic contact region of the cytochrome c molecules. In addition, the prosthetic haem moiety was also shown to provide a significant structural role in terms of relative protein stability under reversed-phase chromatographic conditions. Overall, the results of the present study further demonstrate the utility of interactive modes of chromatography to provide information on physicochemical aspects of protein-surface interactions.