There are conflicting reports concerning the reliability of mass isotopomer distribution analysis (MIDA) for estimating the contribution of gluconeogenesis to total glucose production (f) during [¹³C]glycerol infusion.¹ We have evaluated substrate-induced effects on rate of appearance (R_a) of glycerol and glucose and f during [2-¹³C]glycerol infusion in vivo. Five groups of mice were fasted for 30 h and then infused with [2-¹³C]glycerol at variable rates and variable ¹³C enrichments (group I: 20 µmol · kg¹ · min¹, 99% ¹³C; group II: 60 µmol · kg¹ · min¹, 60% ¹³C; group III: 60 µmol · kg¹ · min¹, 99% ¹³C; group IV: 120 µmol · kg¹ · min¹, 40% ¹³C; or group V: 120 µmol · kg¹ · min¹, 99% ¹³C). The total glycerol R_a increased from ~104 to ~157 and to ~210 µmol · kg¹ · min¹ as the infusion of [2-¹³C]glycerol increased from 20 to 60 and to 120 µmol · kg ¹ · min¹, respectively. As the amount of 99% enriched [2-¹³C]glycerol increased from 20 to 60 and to 120 µmol · kg¹ · min¹ (groups I, III, and V, respectively), plasma glycerol enrichment increased from ~21 to ~42 and to ~57% and the calculated f increased from ~27 to ~56 and to ~87%, respectively. Similar plasma glycerol enrichments were observed in groups I, II, and IV (i.e., ~21-24%), yet f increased from ~27 to ~57 and to ~86% in groups II and IV, respectively. Estimates of absolute gluconeogenesis increased from ~14 to ~33 and ~86 µmol · kg¹ · min¹ as the infusion of [2-¹³C]glycerol increased from 20 to 60 and 120 µmol · kg¹ · min¹. Plausible estimates of f were obtained only under conditions that increased total glycerol R_a ~2-fold (P < 0.001) and increased glucose R_a ~1.5-fold (P < 0.01) above basal. We conclude that in 30-h fasted mice, 1) estimates of f by MIDA with low infusion rates of [2-¹³C]glycerol yield erroneous results and 2) reasonable estimates of f are obtained at glycerol infusion rates that perturb glycerol and glucose metabolism.