The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.