To overcome the problem of insufficient conformational sampling within biomolecular simulations, we have developed a novel Hamiltonian replica exchange molecular dynamics (H-REMD) scheme that uses soft-core interactions between those parts of the system that contribute most to high energy barriers. The advantage of this approach over other H-REMD schemes is the possibility to use a relatively small number of replicas with locally larger differences between the individual Hamiltonians. Because soft-core potentials are almost the same as regular ones at longer distances, most of the interactions between atoms of perturbed parts will only be slightly changed. Rather, the strong repulsion between atoms that are close in space, which in many cases results in high energy barriers, is weakened within higher replicas of our proposed scheme. In addition to the soft-core interactions, we proposed to include multiple replicas using the same Hamiltonian/level of softness. We have tested the new protocol on the GTP and 8-Br-GTP molecules, which are known to have high energy barriers between the anti and syn conformation of the base with respect to the sugar moiety. During two 25 ns MD simulations of both systems the transition from the more stable to the less stable (but still experimentally observed) conformation is not seen at all. Also temperature REMD over 50 replicas for 1 ns did not show any transition at room temperature. On the other hand, more than 20 of such transitions are observed in H-REMD using six replicas (at three different Hamiltonians) during 6.8 ns per replica for GTP and 12 replicas (at six different Hamiltonians) during 8.7 ns per replica for 8-Br-GTP. The large increase in sampling efficiency was obtained from an optimized H-REMD scheme involving soft-core potentials, with multiple simulations using the same level of softness. The optimization of the scheme was performed by fast mimicking [J. Hritz and C. Oostenbrink, J. Chem. Phys. 127, 204104 (2007)]. © 2008 American Institute of Physics.