The isotope dilution method has been widely employed to measure glucose turnover in man. The validity of this technique depends upon several assumptions. First, it is assumed that the selected model of glucose kinetics is valid. Under nonsteady state conditions this has recently been challenged for the most commonly used single compartment model. Secondly, it is assumed that the isotope is metabolized in the same manner as unlabeled glucose. If the isotope loses its label when subject to a substrate cycle (for example, [2-3H]glucose in glucose to glucose 6-phosphate cycling), an overestimate of glucose disposal will result. In contrast, if isotope that has been cleared (for example [6-3H]glucose in hepatic glucogen) is recycled into the systemic circulation, an underestimation of glucose turnover will result. Thirdly, it is assumed that measurement of specific activity is accurate. It has recently been shown that many commercially available tritiated (but not 14C) isotopes contain radioactive nonglucose contaminants which have a slower clearance rate than glucose under conditions of high glucose turnover. This can result in overestimates of specific activity and underestimates of glucose appearance, leading to calculated negative hepatic glucose release. While this problem may be avoided with purified tritiated glucose tracers, a similar problem with stable isotopes of glucose remains to be determined. In summary, the choice of glucose isotope should reflect the probability of substrate cycling and isotope cycling in the experiment in question. Care should be taken to ensure purified isotopes are selected and that the model of glucose kinetics used is valid during the conditions to be studied.