AbstractAdrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4–114 and 4–108, were studied by high‐sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 °C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change δCp = 7.28 ± 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (ΔCp = 4.29 ± 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4–108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.