The pathway of refolding of barnase has been analysed by the protein engineering method using phi plots. The description comprises a folding intermediate, a major transition state (the unfolding transition state) and the fully folded structure. Over 40 mutations have been analysed in the different structural motifs, frequently with several probes in each region. Many of the mutations in this study give phi values for formation of the intermediate of 0, showing that the relevant regions of the structure are as fully unfolded in the intermediate as the unfolded state. Some folding phi values are close to unity, indicating that those regions are fully formed in the intermediate. Even if the data do not report back on a single intermediate but give the averaged properties of a heterogeneous population of sequential or parallel intermediates, then this simplicity of phi data shows that the intermediates tend to have structural features in common. Many phi values are intermediate between those for the unfolded state and the transition state, consistent with either partial structure formation in a single intermediate or a heterogeneous mixture of populations, although the former is more likely. The data are consistent with the intermediate, or collection of intermediates, being on the reaction pathway, rather than side products, because the phi values increase throughout the folding pathway. The main conclusions on the formation of substructure and sequence of folding events from the phi plots are as follows. (1) The major hydrophobic core (core1) begins to form in the intermediate and strengthens in the major transition state. The centre of the core is formed earlier and is stronger in the intermediate and in the transition state than are the edges. (2) Core2 is not formed until after the major transition state. (3) Core3 begins to form in the intermediate and is compact in the transition state. (4) Loop2, loop4 and part of loop1 do not fold until after the major transition state, but the guanosine-binding loop (loop3) is formed in the intermediate and loop5 is partially formed in the intermediate and the transition state. (5) The centre of the beta-sheet is substantially formed in the intermediate, and is fully present in the transition state, but the edges, and associated turns, are definitely weakened.(ABSTRACT TRUNCATED AT 400 WORDS)