A recently introduced tracer, [3,4-(13)C(2)]glucose, was compared to the widely used tracer, [6,6-(2)H(2)]glucose, for measurement of whole-body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4-(13)C(2)]glucose, [6,6-(2)H(2)]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by (13)C NMR (for [3,4-(13)C(2)]glucose) or (1)H and (2)H NMR (for [6,6-(2)H(2)]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 +/- 4 micromol/kg/min. Since (2)H and (13)C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4-(13)C(2)]glucose does not interfere with measurements of (13)C or (2)H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously.