We determined whether overnight inhibition of lipolysis by a long-acting nicotinic acid derivative (acipimox) decreases gluconeogenesis from lactate in NIDDM patients. For this purpose, 250 mg of acipimox or placebo was administered in a double-blind crossover study at 2400, 0400, and 0800 to 8 NIDDM patients (54 ± 4 yr of age, body mass index 29.5 ± 1.3 kg/m2, fasting plasma glucose 11 ± 1 mM). The next morning, total hepatic glucose production (glucose R_a) and gluconeogenesis from lactate were determined using primed, continuous infusions of [3-³H]glucose and [U-¹⁴C]acetate. Glucose and lipid oxidation rates were measured using indirect calorimetry. Mean overnight serum free fatty acid concentrations averaged 242 ± 8 μM after acipimox and 721 ± 30 μM after placebo (P < 0.001). Inhibition of lipolysis decreased lipid oxidation from 33 ± 3 to 22 ± 2 J · kg⁻¹ · min⁻¹ (P < 0.001) and increased carbohydrate oxidation from 15 ± 3 to 23 ± 2 μmol · kg⁻¹ · min⁻¹ (P < 0.005). Gluconeogenesis from lactate decreased by ∼ 40%, from 6.2 ± 0.6 to 3.8 ± 0.5 μmol · kg⁻¹ · min⁻¹ (P < 0.005); lactate oxidation increased from 5.6 ± 0.8 to 7.9 ± 1.1 μmol · kg⁻¹ · min⁻¹ (P < 0.005), with no change in plasma lactate concentrations or total lactate R_d. Fasting plasma glucose concentrations were comparable at 2400 (10.0 ± 1.1 vs. 10.6 ± 1.3 mM, acipimox vs. placebo) and between 0900 and 1000 (10.6 ± 1.3 and 11.3 ± 1.3 mM, respectively). Also, total glucose production rates remained unchanged (14.0 ± 1.2 vs. 14.9 ± 1.3 mol · kg⁻¹ · min⁻¹, respectively). Serum growth hormone (2.8 ± 0.9 vs. 0.5 ± 0.2 μg/l, P < 0.05) and cortisol (444 ± 19 vs. 352 ± 28 nM, P < 0.05) concentrations were higher after acipimox than placebo administration. We conclude that inhibition of lipolysis decreases lipid oxidation and gluconeogenesis from lactate, but fails to lower fasting plasma glucose concentrations and total hepatic glucose production in NIDDM patients.