We investigated the effects of different intravenous (IV) glucose challenges on insulin and proinsulin secretion. On separate occasions, seven normal controls and five obese and five non-insulin-dependent diabetic (NIDDM) subjects each received an IV glucose tolerance test (IVGTT), a hyperglycemic clamp (HY), and a 60-minute, standardized, low-dose, continuous infusion of glucose (CIG) in a randomized fashion. Basal proinsulin concentrations in NIDDM subjects (8.4 ± 5.0 pmol/L) were significantly higher compared with those of normal (1.1 ± 0.2) and obese subjects (1.5 ± 0.4; both P < .05). Basal molar proinsulin: insulin ratio (P:I) was also significantly higher in NIDDM subjects (22% ± 12%) compared with normal (1.0%) and obese subjects (1.6% ± 0.8%; both P < .01). Proinsulin concentrations did not increase significantly in any group during the first 10 minutes of the IV glucose challenges. However, during HY, significant increases in proinsulin concentation occurred after 60 minutes in each group. In normal and obese subjects, IV glucose administration resulted in significant acute increases in insulin concentrations compared with the characteristic blunted response in NIDDM subjects. The P:I ratio in normal and obese subjects did not change in the first 10 minutes after IV glucose administration. However, by the end of HY, the P:I ratio had increased significantly in normal subjects by 1% to 5% ± 2% (P < .05), and in obese subjects by 1% to 5% ± 1% (P < .02). In NIDDM subjects, both HY (19% ± 10% to 27% ± 12%) and IVGTT (18% ± 9% to 43% ± 16%) resulted in a transient increase in the basal P:I ratio by 5 minutes. From 10 minutes until the end of the studies, P:I values were similar to basal values in HY and IVGTT, but decreased significantly during CIG. We conclude that (1) IV glucose administration does not result in first-phase proinsulin secretion in normal or obese subjects; (2) in normal, obese, and NIDDM subjects, sustained or acute IV glucose challenges can produce different proinsulin secretory profiles; (3) prolonged IV glucose challenges can significantly increase the P:I ratio in normal and obese subjects; and (4) a high basal P:I ratio in NIDDM subjects may predict an acute dysfunctional β-cell response to intensive IV glucose challenges.