The contribution of the kidneys to postabsorptive endogenous glucose production is a matter of controversy. To assess whether this could relate to the use of various isotopical methods with different analytical performance capabilities, we measured glucose kinetics in 12 healthy subjects. Blood samples were taken from the femoral artery and the renal vein after 4 h of [6,6-²H₂]glucose infusion (for gas chromatography [GC]/mass spectrometry [MS] analysis), and renal plasma flow was determined with paraaminohippurate. In addition, six subjects received uniformly labeled [¹³C]glucose (for GC/combustion/isotope ratio MS [IRMS]) and [3-³H]glucose (for counting of radioactive disintegrations). Arterial glucose concentrations (means ± SD) were 4.2 ± 0.1 mmol/l, and endogenous glucose production rates using [²H₂]glucose were 2.2 ± 0.1 mg · kg^–1 · min^–1 or 818 ± 50 μmol/min. Dilution of [²H₂]glucose across the kidney was 0.79 ± 1.32%, and renal glucose production (RGP) rates were 27 ± 72 μmol/min. In the six subjects receiving additional tracers, dilutions across the kidney were 2.83 ± 0.72 and 0.54 ± 1.20 (for [U-¹³C]glucose and [3-³H]glucose, respectively, the dilution with [U-¹³C] being higher than that with [²H₂] (P = 0.007). Corresponding RGP values were 144 ± 39 and 43 ± 76 μmol/min for [U-¹³C] and [3-³H], respectively. In conclusion, we found that the highly sensitive [U-¹³C] GC/Combustion/IRMS technique showed consistent dilution of label across the kidney, whereas the less sensitive techniques gave some negative values and smaller RGP rates. Thus, depending on which technique is being used, a fivefold difference in calculated RGP values may be encountered. The methodological variability of our data suggests that extrapolation from regional renal measurements to the whole-body level should be perfumed with caution.