To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ [Lt] Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 °C for Fe2+Rd and 83 °C for Fe3+Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50–70 °C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+Rd, stoichiometric Fe3+/cysteine–ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.