Among the many tracer methods to indirectly estimate gluconeogenesis in humans, the [U-¹³C₆]glucose method as proposed by Tayek and Katz (Am J Physiol Endocrinol Metab 270: E709-E717, 1996; Am J Physiol Endocrinol Metab 272: E476-E484, 1997) has the advantage of being able to simultaneously estimate hepatic glucose output and fractional gluconeogenesis. However, Landau et al. (Landau BR, J Wahren, K Ekberg, SF Previs, D Yang, and H Brunengraber. Am J Physiol Endocrinol Metab 274: E954-E961, 1998) have shown that this method underestimates the rate of gluconeogenesis. The underestimation has been attributed to tracer dilution by other three-carbon substrates and the lack of isotopic steady state. Using a computer simulation of [U-¹³C₆]glucose infusion, we demonstrate that the lack of isotope equilibrium in both the lactate and glucose compartments contributes substantially to the underestimation of gluconeogenesis. [U-¹³C₆]glucose experiments were performed with the addition of a primed constant infusion of [U-¹³C₃]lactate and the delay in M3 glucose equilibrium estimated from the isotopic steady-state value determined by modeling M3 glucose to a single-exponential fit. We found that, even with the addition of [U-¹³C₃]lactate infusion, the M3 glucose enrichment of the last timed sample was ~20% less than the isotopic steady-state value. Thus the lack of isotopic equilibrium of the glucose compartment potentially accounts for 20% of the underestimation of gluconeogenesis. The underestimation of gluconeogenesis using [U-¹³C₆]glucose without the additional infusion of [U-¹³C₃]lactate in previous publications is expected to be even greater because of the lack of isotope equilibrium in both the lactate and glucose compartments. These findings are consistent with the results from our computer simulation.