In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.