In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.