Concentrations of human C-peptide, IRI (immunoreactive insulin) and glucose were determined during oral glucose tolerance test (1.75 g glucose/kg ideal body weight) in 14 normal persons (N), 9 maturity-onset diabetics (DI) and 10 insulin-requiring diabetics (DII) never treated with insulin and in 3 formerly insulin treated diabetics. The mean fasting levels of C-peptide and IRI in the first three groups were: N: 0.37 ± 0.02 nM and 0.048 ± 0.009 nM, DI: 0.86 ± 0.17 nM and 0.11 ± 0.029 nM, DII: 0.37 ± 0.04 nM and 0.063 ± 0.009 nM. One hour after oral glucose ingestion, the respective values increased to: N: 2.53 ± 0.20 nM and 0.52 ± 0.077 nM, DI: 2.49 ± 0.31 nM and 0.49 ± 0.11 nM, DII: 0.49 ± 0.05 nM and 0.11 ± 0.014 nM. Although secreted from the pancreas in equimolar concentrations, the molar ratio of C-peptide to insulin in peripheral blood was about 7 in the fasting state, falling to about 5 in the glucose stimulated condition. Maturity-onset diabetics had higher fasting levels of C-peptide than normal subjects, in agreement with the IRI levels. Three patients previously treated with insulin and having insulin antibodies showed C-peptide responses significantly below the normal range. In one of these patients, the test was repeated 9 months later when the insulin antibodies had disappeared, and the C-peptide response observed at that time was much higher. It is suggested that insulin antibodies cause an impaired IRI — and consequently C-peptide response — by constantly removing insulin from the granules in the B-cell. In normal humans the peripheral C-peptide responses to the oral glucose load showed less relative variation than do the insulin responses. Therefore, a radioimmunoassay for C-peptide in addition to the assay for insulin will provide supplementary information on insulin secretion.