The charge transfer band at 695 nm in the spectrum of ferri-cytochrome c is highly asymmetric indicating conformational heterogeneity due to coexistence of different conformational substates. We have measured the respective band profile of horse heart ferri-cytochrome c as a function of temperature between 100 and 600C and found that the well known decrease of the absorptivity is wavenumber dependent and exhibits a biphasic behavior. This indicates that the underlying conformational substates differ in their thermodynamic stability with respect to the structural changes associated with the disappearance of the 695 nm band which eventually (at high temperatures) involve the replacement of M80 by a nearby lysine residue. Our data further indicate that the thermal unfolding process involves two structurally different intermediate states.