Excessive hepatic glucose output is an important factor in the fasting hyperglycemia of non-insulindependent diabetes mellitus (NIDDM). To determine the relative contributions of gluconeogenesis and glycogenolysis in a quantitative manner, we applied a new isotopic approach, using infusions of [6-³H]glucose and [2-¹⁴C]acetate to trace overall hepatic glucose output and phosphoenolpyruvate gluconeogenesis in 14 postabsorptive NIDDM subjects and in 9 nondiabetic volunteers of similar age and weight. Overall hepatic glucose output was increased nearly twofold in the NIDDM subjects (22.7 ± 1.0 vs. 12.0 ± 0.6 μmol · kg⁻¹ · min⁻¹ in the nondiabetic volunteers, P < .001); phosphoenolpyruvate gluconeogenesis was increased more than threefold in the NIDDM subjects (12.7 ± 1.4 vs. 3.6 ± 0.4 μmol kg⁻¹ min⁻¹ in the nondiabetic subjects, P < .001) and was accompanied by increased plasma lactate, alanine, and glucagon concentrations (all P < .05). The increased phosphoenolpyruvate gluconeogenesis accounted for 89 ± 6% of the increase in overall hepatic glucose output in the NIDDM subjects and was significantly correlated with the fasting plasma glucose concentrations (r = .67, P < .01). Glycogenolysis, calculated as the difference between overall hepatic glucose output and phosphoenolpyruvate gluconeogenesis, was not significantly different in the NIDDM subjects (9.9 ± 0.06 μmol · kg⁻¹ · min⁻¹) and the nondiabetic volunteers (8.4 ± 0.3 μmol kg⁻¹ · min⁻¹). We conclude that increased gluconeogenesis is the predominant mechanism responsible for increased hepatic glucose output in NIDDM.