AbstractIn the present work, we have thermodynamically characterized the thermally induced unfolding of 20 variants of bovine pancreatic ribonuclease A (RNase A) to experimentally describe the residues and the regions that are critical for the stability of the enzyme. The achieved results, complemented with previous studies by our group, allowed us to define the significance of the two hydrophobic nuclei present in the RNase A structure, as well as the contribution of the participating residues within each nucleus, to the global enzyme stability. We propose a structural model for the major and the minor hydrophobic nuclei of RNase A. The major nucleus is composite and located in the cavity delimited by α‐helices 1 and 3, and the β‐sheet that is formed by strands 2, 3, 5, and 6. It consists of a central tight packed part constituted by residues Phe8, Met13, Val54, Val57, Ile106, Val108, and Phe120. This central part is surrounded by a layer formed by residues Val63, Tyr73, Met79, Ile107, Val116, and Val118. The minor nucleus, although less complex, is also constituted by a tight packing that involves the side chains of residues Tyr25, Met29, Met30, Leu35, Phe46, and Tyr97, which fill the cavity that originates the β‐sheet formed by β‐strands 1, 4, and 5 together with α‐helix2. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1038–1047, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com