The development of a simple microscale solid phase screening RIA and improved methods for cell cloning has lead to the establishment of 2 prohormone- and species-specific mouse monoclonal antibodies against human proinsulin (HPI). These antibodies react to determinant(s) only expressed on the HPI molecule. In addition, 11 rat monoclonal antibodies were generated which react with both human C-peptide (HCP) and HPI. All 11 rat antibodies recognize a very similar antigenic determinant in the C-peptide that appears to be made up of residues 40-45 and 57-63, which are probably brought into close proximity by a beta-turn near the center of the connecting segment. The identical behavior of both HPI-specific mouse antibodies in competition experiments indicates that the antigenic structure recognized in proinsulin might be the same for both antibodies. This structure could not be regenerated by mixing equimolar amounts of human insulin and C-peptide, including the chemically synthesized complete proinsulin connecting segment (Arg X Arg X HCP X Lys X Arg), which contains the entire sequence removed from proinsulin in the conversion to mature insulin. Indirect evidence is provided that a HPI molecule simultaneously can bind a C-peptide-directed rat antibody and a HPI-specific mouse antibody when the first antibody is presented to HPI in solution phase. However, when HPI is immobilized on a plastic surface, the binding of 1 type of antibody completely blocks the binding of the other. These antibodies provide useful new tools for studying the biosynthesis and 3-dimensional structure of HPI and HCP.