Insulin and C-peptide responses to 0.5 g kg-1 intravenous glucose and 1.0 mg glucagon were studied in 34 healthy subjects (age 19-78 years, mean 45). Fasting blood glucose (r = 0.59; p < 0.001) and glycosylated haemoglobin (r = 0.61; p < 0.001) increased with age, but not the initial C-peptide and insulin responses to the glucose infusion. However, the C-peptide response at 70 min (r = 0.36; p < 0.05), 80 min (r = 0.41; p < 0.05), and 90 min (r = 0.46; p < 0.01) after the glucose infusion correlated with age as well as both insulin (r = 0.42; p < 0.05) and C-peptide (r = 0.45; p < 0.05) responses to the glucagon injection. Reproducibility of insulin and C-peptide responses was evaluated by duplicate tests, separated 2-143 days in time, in 10 healthy subjects (age 19-48 years, mean 32 years) showing no significant differences in median within-subject variation between the initial (1 + 3 min) or overall (0-90 min area under curve) insulin (24% and 17%, respectively) and C-peptide (15% and 14%, respectively) responses to glucose, while the within-subject variation for the fasting values and the response to glucagon was higher (p < 0.05) for insulin (47% and 32%, respectively) than C-peptide (13% and 14%, respectively). Between-subject variation was also lower (p < 0.001) for C-peptide than for insulin. Thus, C-peptide measurements in healthy subjects are more reproducible than insulin measurements in determination of beta-cell function.