An improved synthesis of human C-peptide is described. Five fragments: 33-39, 40-46, 47-49, 50-54 and 55-63 were used in the total synthesis. In the fully protected C-peptide the N-terminal alpha-amino function was blocked by a benzyloxycarbonyl group and the carboxyl and serine hydroxyl functions were blocked by t-butyl protection. The latter protecting groups were removed by trifluoroacetic acid to obtain N-alpha-benzyloxycarbonyl human C-peptide which, on catalytic hydrogenation, yielded human C-peptide. The immunoreactivity of the prepared human C-peptide was tested and found to deviate slightly from the human C-peptide synthesized earlier by another route. When tested in the immunoassay, human pancreatic extracts containing natural C-peptide (or fragments thereof) showed dilution patterns identical to that of the new synthetic C-peptide but different from that of the previously synthesized batch of C-peptide. The possible explantation for the observed differences in the immunoreactivity is discussed.