Insulin and C-peptide secretion rates have been measured and compared in 12 nondiabetic subjects to characterize the glucose stimulus-response of B cell secretion in man. On three different days, glucose concentrations were clamped for 150 minutes at 7.5, 10, and 15 mmol/L, respectively. Plasma samples taken during the clamps were assayed for C-peptide and insulin. C-peptide secretion rates were estimated by the technique of deconvolution. Model-based estimation of insulin secretion rates from insulin concentrations yielded concordant results. In response to glucose, C-peptide concentrations rose less quickly than did insulin concentrations, but the estimated first- and second-phase secretion rates were similar when assessed from either the C-peptide or insulin concentrations. First-phase secretion peaks were larger than inspection of the plasma concentration data might suggest, with median values of 1.3, 2.0, and 2.9 nmol/min for C-peptide in response to 7.5, 10, and 15 mmol/L glucose clamp levels, respectively. The second-phase reached steady state by 90 to 120 minutes, with median C-peptide secretion rates of 0.31, 0.56, and 0.85 nmol/min after 120 minutes at 7.5, 10, and 15 mmol/L, respectively. The slopes of the curves of steady-state insulin and C-peptide secretion rates v the four glucose levels (basal plus the three clamp levels) were maximally steep between 7.5 and 10 mmol/L in the majority of subjects, consistent with in vitro sigmoidal responses. A characterization of the secretory response of the B cell of normal humans at different glucose concentrations has been obtained. With appropriate models, insulin secretion rates may be estimated from either plasma insulin or C-peptide concentration data.