BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration.