A serious limitation in the study of protein folding reactions resides in the lack of experimental methods to measure the absolute height of the free energy barrier. This is particularly unfortunate given that if folding barriers are small, as theory predicts, it might be possible to resolve folding mechanisms directly. Here we explore the performance of a recently developed method to extract folding barriers from equilibrium differential scanning calorimetry (DSC) experiments. To test the method, we compare the thermodynamic barrier heights for 15 proteins obtained from available DSC data with the folding rates measured in kinetic experiments. The correlation between these two parameters is very good (r2 = 0.9) and has a slope consistent with the same energy scale. These results confirm that it is possible to measure free energy barriers for natural proteins from equilibrium DSC experiments. Furthermore, the measured barrier heights are small (<8 RT), in general, and marginal or nonexisting for fast-folding proteins. Copyright © 2005 American Chemical Society.