To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (Delta G(HX)) and the kinetic unfolding and refolding rates (k(op) and k(cl)) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 x 10(-6) s(-1) and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the beta-barrel, including mutually H-bonding residues in the beta 4 and beta 5 strands, a part of the beta 3 strand that H-bonds to beta 5, and residues at the N-terminus of the alpha2 helix that is capped by beta 5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native alpha2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded beta1-beta2-beta 3 meander, completing the native beta-barrel, plus an adjacent part of the alpha1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.