Empirical analysis of the primary structures of 10 mammalian C-peptides has indicated a conservation of conformation. The positioning of the C-peptide in the proinsulin molecule is essentially defined by the proposed secondary structure, the covalent connection to the A1 and B30 residues of insulin and the requirement that C-peptide lies against the exposed surface of the insulin hexamer, allowing a three-dimensional structure for proinsulin to be predicted. Conserved residues in the C-peptide are proximate to residues in insulin that are also conserved, suggesting that interactions between these residues are highly probable. Residues in insulin thought to be important for biological activity are principally those that interact with the C-peptide residues. The role of the C-peptide region in proinsulin in preventing expression of insulin activity and for nucleation of the folding of the prohormone are discussed.