Anaplerotic enzymes, such as pyruvate carboxylase or malic enzyme, catalyze reactions that fill up the pools of the citric acid cycle (CAC), thereby increasing the total mass of CAC intermediates. Relative anaplerosis (y) denotes the ratio of anaplerotic flux to the flux catalyzed by citrate synthase. We examine conventional methods [C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey. J. Biol. Chem. 263:6964-6971, 1988; C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H987-H995, 1990] of measurement of y using ¹³C-labeled precursors and analysis of [¹³C]glutamate labeling by nuclear magnetic resonance (NMR) spectroscopy. Through mathematical analysis and computer simulation, we show that isotopic enrichment of the pool of pyruvate that is substrate for anaplerosis will severely decrease the accuracy of estimates of y made with conventional methods no matter how small the mass of the pool of pyruvate. Suppose that the recycling parameter R denotes the fraction of molecules of pyruvate that contain carbons derived from intermediates of the CAC. Each means of estimation of relative anaplerosis in the peer-reviewed literature assumes that R = 0, although this assumption has not been confirmed by experiment. We show that conventional formulas, using either fractional enrichments of carbons or isotopomer analysis, actually estimate at most y · (1  R) instead of y during administration of [2-¹³C]acetate and unlabeled pyruvate. Using a new formula for estimation of y, we recalculate values of y from the literature and find them ~50% too low. We assume that all anaplerosis is via pyruvate and that the difference in isotopic enrichment between cytosolic and mitochondrial malate is negligible.