A sensitive and specific radioimmunoassay for rat serum C-peptide (RCP) has been developed and validated using a guinea pig anti-rat C-peptide antibody to synthetic rat C-peptide. Negligible crossreactivity (< 0.01%) to human proinsulin was observed, whereas human insulin, human pancreatic polypeptide (hPP), porcine insulin, porcine C-peptide, bovine insulin, rat insulin, porcine-PP, and glucagon, respectively, did not produce measurable displacement of RCP tracer. Human C-peptide even in a supraphysiological concentration range crossreacted poorly (< 0.1%). The sensitivity limit of the assay calculated at +/- 3 standard deviations was 24.2pM (0.07 ng/mL). RCP standard concentrations ranged from 25-1600pM. The intraassay- and between assay-coefficient of variations (CV) were 3.5-6.1% and 4.1-9.5%, respectively. The mean percentage recovery of RCP added to rat serum samples was 100.8 +/- 2%. Serum volume dilution from 25 to 100 microL did not significantly alter the expected RCP level. Migration of rat serum C-peptide and that of synthetic RCP were identical in a Sephadex G-50 chromatographic analysis. The mean fasting and postprandial plasma RCP levels in normal rats were 102 +/- 15 pM and 485 +/- 75pM, respectively. RCP levels following intravenous glucose tolerance test in diabetic and nondiabetic rats were consistent with expected patterns. In conclusion, we have developed and validated a rat C-peptide assay that is sensitive, simple, and specific for RCP in serum. The assay provides a reliable tool for studies of diabetes using rodent animal models.