Increased gluconeogenesis has been suggested to account for all of the increase in basal glucose production in patients with non-insulin-dependent diabetes mellitus (NIDDM). We studied the effect of inhibition of gluconeogenesis with ethanol on total hepatic glucose output (HGO) in patients with NIDDM. Fourteen patients with NIDDM (mean ± SE age 61 ± 2 yr, fasting plasma glucose 11.4 ± 0.8 mM; body mass index 27 ± 1 kg/m2) were studied twice after an overnight fast, once during ethanol administration (blood ethanol ∼12 mM) and once during saline administration. Total HGO rate was measured with [3H]glucose. Inhibition of gluconeogenesis by ethanol was followed qualitatively with [U-14C]lactate (n = 8) and [U-14C]glycerol (n = 6) as tracers. Ethanol inhibited gluconeogenesis from lactate by 71 ± 5% (0.5 ± 0.2 vs. 1.8 ± 0.1 μmol glucose · kg−1 · min−1, 240–300 min, P < 0.001; ethanol vs. saline, P < 0.001) and from glycerol by 65 ± 6% (0.8 ± 0.2 vs. 2.3 ± 0.6 μmol glucose · kg · min−1, P < 0.001). Total HGO rate remained unchanged and averaged 12.8 ± 1.8 and 11.8 ± 2.1 μmol · kg−1 · min−1 in the saline and ethanol studies, respectively (NS). We concluded that inhibition of gluconeogenesis by ethanol does not decrease total HGO in patients with NIDDM. Our results suggest the existence of a regulatory mechanism in the liver that maintains constant total HGO despite inhibition of gluconeogenesis.