I evaluated the between-method variation of C-peptide determined with C-peptide radioimmunoassay kit reagents from Mallinckrodt, Behring, Diagnostic Products Corp., and Immuno Nuclear Corp., and antisera from Novo (M1230, M1221, and K6) and Cambridge Medical Diagnostics (299-029P). C-peptide concentrations corresponding to 0.6 nmol/L (as determined with M1230) ranged from 0.54 (by Immuno Nuclear Corp.) to 1.06 nmol/L (by M1221). Furthermore, pretreating plasma specimens with Polyethylene Glycol 6000 variably decreased the results. Several factors may contribute to the between-method variation of C-peptide analyses: differences in the specificity of antisera, the type of C-peptide preparation used as standard and tracer, liability of the immunoassay to nonspecific interference, and possibly the heterogeneity of C-peptide immunoreactivity in the specimens.