Antisera to the C-peptide of human proinsulin were obtained by immunizing guinea pigs with synthetic benzyloxycarbonyl-C-peptide conjugated to human albumin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In three series of 10, the animals were injected with C-peptide conjugated to albumin in the molar ratio of 23 : 1, 15 : 1, and 4 : 1, respectively. Antibodies to human C-peptide were present in all the surviving 25 animals. Fifteen of the antisera were suitable for measuring C-peptide concentrations lower than 0.10 pmol/ml. The antisera demonstrated an increasing immunogenicity with increasing molar ratio of C-peptide to albumin in the conjugate. In the fourth series, ten guinea pigs immunized with benzyloxycarbonyl-C-peptide ionically bound to QAE-Sephadex A-25 did not produce detectable antibodies to C-peptide. A qualitative evaluation of the radioimmunoassay by use of the antiserum with the highest titer and sensitivity, "M 1230", revealed a mean intra-assay and inter-assay coefficient of variance of 3.2 and 9.6%, respectively.