The acute sensitivity to conformation exhibited by amide hydrogen exchange reactivity provides a valuable test for the physical accuracy of model ensembles developed to represent the Boltzmann distribution of the protein native state. A number of molecular dynamics studies of ubiquitin have predicted a well-populated transition in the tight turn immediately preceding the primary site of proteasome-directed polyubiquitylation Lys 48. Amide exchange reactivity analysis demonstrates that this transition is 10(3)-fold rarer than these predictions. More strikingly, for the most populated novel conformational basin predicted from a recent 1 ms MD simulation of bovine pancreatic trypsin inhibitor (at 13% of total), experimental hydrogen exchange data indicates a population below 10(-6). The most sophisticated efforts to directly incorporate experimental constraints into the derivation of model protein ensembles have been applied to ubiquitin, as illustrated by three recently deposited studies (PDB codes 2NR2, 2K39 and 2KOX2K392KOX). Utilizing the extensive set of experimental NOE constraints, each of these three ensembles yields a modestly more accurate prediction of the exchange rates for the highly exposed amides than does a standard unconstrained molecular simulation. However, for the less frequently exposed amide hydrogens, the 2NR2 ensemble offers no improvement in rate predictions as compared to the unconstrained MD ensemble. The other two NMR-constrained ensembles performed markedly worse, either underestimating (2KOX) or overestimating (2K39) the extent of conformational diversity.