The effect of alteration in isocratic mobile phase constituents, composition of sample solution, flow-rate and column temperature on the reversed-phase chromatographic behaviour of beta-endorphin was investigated. Beta-Endorphin was shown to be particularly sensitive to the concentration of organic modifier within the mobile phase. The relative contact area of beta-endorphin was demonstrated to be less than that of the much smaller molecule, gamma-endorphin, indicating that beta-endorphin is in a folded form under the mobile phase conditions utilised. Buffer molarity and pH were implicated in the conformational transition of beta-endorphin. In addition, the micro-environment of beta-endorphin prior to its injection onto the high-performance liquid chromatographic (HPLC) column is crucial to its chromatographic behaviour. Manipulation of the sample solvent environment produced reversible conformational modifications ultimately resulting in asymmetric and even split peaks. This phenomenon was more clearly seen when altering HPLC flow-rate. Elevation of HPLC column temperature provided additional evidence of structural change in beta-endorphin, with further conformational forms of this molecule being observed at higher temperatures. This work suggests that the chromatography of beta-endorphin involves a complex mechanism of separation which cannot be adequately explained by the two-state model of kinetic processes.