To determine whether [6-3H]glucose could be used to quantitatively estimate the rate of plasma glucose conversion to plasma lactate, we compared the relative transfer of [3H] and [14C]plasma glucose to plasma lactate in nine postabsorptive anesthetized rats infused to isotopic steady state with [6-3H]glucose and [6-14C]glucose. Glucose turnover (mumol/kg/min) measured with [6-3H]glucose (29.4 +/- 1.7) and [6-14C]glucose (25.0 +/- 1.5), Cori cycle activity (4.35 +/- 0.79), and the percentage of plasma lactate derived from plasma glucose calculated from 14C-specific activities (59.8 +/- 5.8) were all comparable to previously reported values for the overnight fasted rat. Although the percentage of plasma lactate derived from plasma glucose calculated from 3H-specific activities (30.0 +/- 3.3) was only half that calculated from 14C-specific activities (P less than 0.001), the 3H:14C ratios were constant over a wide range of percentage of lactate derived from glucose (r = 0.95, P less than 0.001). We conclude that 3H-specific activity ratios of plasma lactate to plasma glucose cannot be directly used as a quantitative estimate of the percentage of plasma lactate derived from plasma glucose; however, correction for the apparent constant proportionality of detritiation of lactate relative to 14C loss could permit use of [6-3H]glucose for this purpose.