Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.