To test whether gluconeogenesis is increased in non-insulin-dependent diabetic (NIDDM) patients we infused (post-absorptive state) healthy subjects and NIDDM patients with [6,6-²H₂]glucose (150 min) and [3-¹³C]lactate (6 h). Liver glutamine was sampled with phenylacetate and its labelling pattern determined (mass spectrometry) after purification of the glutamine moiety of urinary phenylacetylglutamine. After correction for ¹³CO₂ re-incorporation (control test with NaH¹³CO₃ infusion) this pattern was used to calculate the dilution factor (F) in the hepatic oxaloacetate pool and fluxes through liver Krebs cycle. NIDDM patients had increased lactate turnover rates (16.18 ± 0.92 vs 12.14 ± 0.60 μmol · kg⁻¹· min⁻¹, p < 0.01) and a moderate rise in glucose production (EGP) (15.39 ± 0.87 vs 12.52 ± 0.28 μmol · kg⁻¹· min⁻¹, p = 0.047). Uncorrected contributions of gluconeogenesis to EGP were 31 ± 3 % (control subjects) and 17 ± 2 % (NIDDM patients). F was comparable (1.34 ± 0.02 and 1.39 ± 0.09, respectively) and the corrected percent and absolute contributions of gluconeogenesis were not increased in NIDDM (25 ± 3 % and 3.8 ± 0.5 μmol · kg⁻¹· min⁻¹) compared to control subjects (41 ± 3 % and 5.1 ± 0.4 μmol · kg⁻¹· min⁻¹). The calculated pyruvate carboxylase over pyruvate dehydrogenase activity ratio was comparable (12.1 ± 2.6 vs 11.2 ± 1.4). Lastly hepatic fatty oxidation, as estimated by the model, was not increased in NIDDM (1.8 ± 0.4 vs 1.6 ± 0.1 μmol · kg⁻¹· min⁻¹). In conclusion, in the patients studied we found no evidence of increased hepatic fatty oxidation, or, despite the increased lactate turnover rate, an increased gluconeogenesis. [Diabetologia (1998) 41: 212–220]