AbstractTo investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well‐folded RNases H. Stability curves, based on CD‐monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (ΔCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low ΔCp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.