We evaluated the reproducibility of different estimates of endogenous insulin secretion in 30 patients with non-insulin-dependent diabetes mellitus (NIDDM). Fasting blood glucose concentration was similar on the 2 days of study. The coefficients of variation of fasting plasma C-peptide, plasma C-peptide 6 min after the injection of 1 mg i.v. glucagon, and the increment in plasma C-peptide after glucagon were 16.0, 14.8 and 24.1%, respectively. The coefficients of variation of the corresponding plasma insulin values were 19.2, 24.8, and 34.8%, respectively. The coefficient of variation of 24-h urinary C-peptide excretion was 22.1%. Because fasting plasma C-peptide correlated closely with plasma C-peptide 6 min after glucagon (test 1: r = .70, P less than .01; test 2: r = .76, P less than .01), it seems that these two values can be used equally well as assessment of beta-cell function in NIDDM. In conclusion, fasting plasma insulin, fasting plasma C-peptide, and plasma C-peptide 6 min after glucagon stimulation showed a similar and acceptable degree of reproducibility. Plasma insulin 6 min after glucagon and increments in plasma insulin and C-peptide, as well as urinary C-peptide, seem to be less reproducible.