Several solved X-ray structures and an NMR ensemble of the dimeric λ Cro protein markedly differ from one another, leaving its dominant solution conformation in question. Recently performed replica exchange molecular dynamics simulations starting from crystal structure coordinates sample a range of conformational space that correlates well with the X-ray structures but differs notably from the NMR models. To further investigate the latter observation, this study focuses on additional simulation beginning from an NMR model. Network analysis and a free energy surface show that the trajectory immediately converges to a region of conformational space in agreement with the previously run simulations and the crystal structures, but they suggest that the NMR models are not stable solution conformations. Insufficient intersubunit nuclear Overhauser enhancement restraints and the absence of electrostatics in the structure calculation may have compromised the global conformation of the NMR models. © 2012 Wiley Periodicals, Inc.