During ribonuclease S (RNase S) refolding, two peptide fragments recognize each other, and bind together to form a refolding intermediate which slowly converts to the native state. We have characterized this refolding intermediate using absorbance, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopies. These techniques reveal significant amounts of both secondary and tertiary structure; the intermediate differs from a molten globule in being packed and native-like, but it resembles a molten globule in having no near-ultraviolet (UV) CD spectrum. Final refolding is slow and accompanies proline isomerization. The results show that at least two separate stages are observed in the formation of the tertiary structure of RNaseS.