The objective of the study was to evaluate the effect of insulin treatment on insulin secretion in patients with non-insulin-dependent diabetes mellitus (NIDDM). Ten patients with NIDDM were first investigated while still taking oral hypoglycemic agents, and then randomized to a crossover study with two eight-week periods of insulin treatment (oral treatment having been stopped) given either as mainly intermediate-acting insulin twice daily (2-dose) or as preprandial regular insulin and intermediate-acting insulin at bedtime (4-dose). In the patients treated with oral agents the 24-h C-peptide area under the curve was similar to that in the controls, but the profile was different with a rise at breakfast but with almost absent meal peaks during the rest of the day. Insulin treatment improved glycemic control markedly, lowered urinary C-peptide excretion and the serum C-peptide concentrations being reduced by more than 50%. The shape of the C-peptide profiles was unaltered and there were no significant differences between the two insulin regimens. The decrease in serum C-peptide concentration during insulin treatment correlated with the change in blood glucose. Fasting serum C-peptide concentrations correlated closely with the 24-h C-peptide area under the curve. In conclusion, insulin treatment of NIDDM patients with secondary failure to oral agents greatly reduces the insulin secretion, probably owing to the reduction in blood glucose.