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.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 ⋅ kg⁻¹ ⋅ min⁻¹, 99% ¹³C;group II: 60 μmol ⋅ kg⁻¹ ⋅ min⁻¹, 60% ¹³C;group III: 60 μmol ⋅ kg⁻¹ ⋅ min⁻¹, 99% ¹³C;group IV: 120 μmol ⋅ kg⁻¹ ⋅ min⁻¹, 40% ¹³C; orgroup V: 120 μmol ⋅ kg⁻¹ ⋅ min⁻¹, 99% ¹³C). The total glycerol R_a increased from ∼104 to ∼157 and to ∼210 μmol ⋅ kg⁻¹ ⋅ min⁻¹as the infusion of [2-¹³C]glycerol increased from 20 to 60 and to 120 μmol ⋅ kg^{− 1} ⋅ min⁻¹, respectively. As the amount of 99% enriched [2-¹³C]glycerol increased from 20 to 60 and to 120 μmol ⋅ kg⁻¹ ⋅ min⁻¹(groups I,III, andV, 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 ingroups I,II, andIV (i.e., ∼21–24%), yet f increased from ∼27 to ∼57 and to ∼86% ingroups II andIV, respectively. Estimates of absolute gluconeogenesis increased from ∼14 to ∼33 and ∼86 μmol ⋅ kg⁻¹ ⋅ min⁻¹as the infusion of [2-¹³C]glycerol increased from 20 to 60 and 120 μmol ⋅ kg⁻¹ ⋅ min⁻¹. 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.