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Monoallelic point mutations in the cytosolic, NADP+-dependent enzyme isocitrate dehydrogenase 1 (IDH1) are associated with elevated production of the oncometabolite 2-hydroxyglutarate (2-HG) in multiple cancers. Production of 2-HG by mutant R132H IDH1 requires activity of a retained wild-type (WT) allele, suggesting possible channeling of α-ketoglutarate and NADPH in WT/R132H IDH1 heterodimers [1]. We report a mechanistic characterization of IDH1 2-HG production using a combination of mathematical modeling, in vivo kinetic measurements, and diffusion calculations. Numerical simulation of a biochemically realistic mathematical model of the IDH1 heterodimer indicated that substrate channeling would have a large impact on the dynamics of 2-HG generation. We then examined conversion of 13C labeled glucose and 13C labeled glutamine into labeled 2-HG in HT1080 cells and found that the dynamics are nearly identical in both cases. This result provides strong evidence against channeling because the production of 2-HG from glucose, but not glutamine, is dependent on WT IDH1. Calculations of substrate diffusion between the WT and mutant active sites of both isolated and agglomerated IDH1 heterodimers further support the absence of channeling. We propose instead that 2-HG production is sensitive to the global availability of substrates in the appropriate intracellular compartment, consistent with prior observations that 2-HG production by structurally analogous mutants of the mitochondrial isoform IDH2 is not WT-dependent [1]. Building on this observation, we report a strategy for inferring compartment-specific NADPH/NADP+ ratios using our model and steady-state measurements of 2-HG production by native IDH1 and by IDH1 artificially targeted to the mitochondrion.[1] Ward, P.S., C. Lu, J.R. Cross, O. Abdel-Wahab, R.L. Levine, G.K. Schwartz, and C.B. Thompson. 2013. J. Biol. Chem. 288: 3804-3815