Antigenic determinants recognized by human proinsulin (HPI)-specific monoclonal antibodies (Mabs) and Mabs crossreacting with free human C-peptide (HCP) were mapped by using various forms of purified, partially converted HPI intermediates. Two HPI-specific mouse Mabs (GS-4G9 and GS-9A8) reacted with the same antigenic determinant, GS, which was localized to the site of linkage of the B-chain to the C-peptide (Arg-Arg) at positions 31–32. These antibodies bind with equal efficiency to C65-A1 split proinsulin and to intact HPI. The binding of C32-C33 split proinsulin is markedly reduced. A rat Mab (GN-VIIB6), which crossreacts with free HCP in addition to HPI, reacted similarly with various HPI intermediates as it had with the corresponding synthetic HCP fragments, as previously reported (see ref. 9). This determinant (GN) is a three-dimensional structure composed of residues located in two separate regions in the C-peptide segment (positions 40–45 and 57–63). Reduced, carboxymethylated HPI retains the GN-determinant, whereas all insulin-like immunoreactivity identified with a conventional guinea pig insulin antiserum is completely lost. The binding of the two GS Mabs to the denatured HPI was reduced by 40–50% compared with intact HPI. It is concluded that the strong GN-determinant can readily form in the C-peptide segment of HPI, independently of the presence of ordered structure in the insulin moiety. A predicted β-turn at position 47–50 may play an important role in bringing N- and C-terminal regions of the C-peptide segment into close proximity. The spontaneous formation of the GN-determinant could thus be a crucial step in the formation of the correct disulfide bonds in more distant parts of the newly synthesized HPI-polypeptide chain