In order to further characterise the role of conformation in the retention behaviour of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four different insulins have been studied as a function of temperature (over the range 5-85°C) and column residence time (over the range 10-60 min). The role of the ligand structure was also investigated by comparing results obtained with a n-octadecyl (C18) and a n-butyl (C4) ligand immobilised to the same porous silica. Comparative structure-retention-stability relationships were determined from an examination of the influence of temperature on a number of chromatographic parameters including the chromatographic contact area, the affinity constant and the experimental band width. The results demonstrated that variations in temperature can be used to affect significant changes in selectivity between the different insulins despite their very high degree of sequence homology. These observations have permitted specific amino acid residues, and in particular those residues encompassing the region A8-A10, to be proposed to be directly involved in the chromatographic contact area of the insulin molecules. Overall, the analysis of the changes in various chromatographic parameters in response to variation of the amino acid sequence, temperature and other experimental parameters provides a powerful tool to elucidate the structural basis for the interfacial stability and the role of conformation on the retention behaviour of polypeptides and proteins in RP-HPLC.