In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data.