C-peptide is a polypeptide originating from proinsulin after its cleavage in the B-cell. It is secreted equimolarly with the other cleavage product, insulin, into the portal circulation. Only a minimal fraction of C-peptide is extracted by the liver; it is supposed to be mainly removed by the kidney. A small, constant proportion is excreted in the urine. C-peptide is measured in serum and urine by radioimmunoassay. The major sources of error of the assay are related to standard, tracer, antiserum specificity and reactivity with proinsulin, and to degradation of C-peptide. Many secretagogues are used to evaluate the B-cell function, of which glucagon is the most simple. Clinical applications of C-peptide include differentiating between endogenous and exogenous hyperinsulinism and establishing the need of insulin therapy in diabetic patients already treated with insulin. The primary value of C-peptide, however, is in clinical research, where it offers a unique opportunity to follow the B-cell secretion in diabetic subjects and to evaluate the difference that various factors may exert on its activity.