Fasting and postprandial plasma glucose, free fatty acid (FFA), lactate, and insulin concentrations were measured at hourly intervals for 24 h in 27 nonobese individuals—9 with normal glucose tolerance, 9 with mild non-insulin-dependent diabetes mellitus (NIDDM, fasting plasma glucose < 175 mg/dl), and 9 with severe NIDDM (fasting plasma glucose > 250 mg/dl). In addition, hepatic glucose production (HGP) was measured from midnight to 0800 in normal individuals and patients with severe NIDDM. Plasma glucose concentration was highest in patients with severe NIDDM, lowest in those with normal glucose tolerance, and intermediate in those with mild NIDDM (two-way ANOVA, P < .001). Variations in plasma FFA and lactate levels of the three groups were qualitatively similar, with lowest concentrations seen in normal individuals, intermediate levels in the group with mild NIDDM, and the highest concentration in those with severe NIDDM (two-way ANOVA, P < .001). Of particular interest was the observation that plasma FFA concentrations were dramatically elevated from midnight to 0800 in patients with severe NIDDM. The 24-h insulin response was significantly increased in patients with mild NIDDM, with comparable values seen in the other two groups. Values for HGP fell progressively throughout the night in normal individuals and patients with severe NIDDM, despite a concomitant decline in plasma glucose and insulin levels. Although the magnitude of the fall in HGP was greater in NIDDM, the absolute value was significantly (P < .001) greater than normal throughout the period of observation. These results demonstrate that there are differences in substrate level between individuals with normal glucose tolerance and patients with NIDDM and differing degrees of glucose intolerance, unrelated to ambient insulin level, and these changes persist over 24 h.