We present gas chromatographic/mass spectrometric and gas chromatographic/isotope ratio mass spectrometric assays of the 13C enrichment of plasma urea converted to its dimethylaminomethylene derivative. The limits of sensitivity of the two techniques are 0.2% and 0.02%, respectively. The techniques were tested in rats and humans infused with (13C)urea or (3-13C)lactate. (13C)Urea enrichment during the infusion of (3-13 C)lactate in humans was not detectable by gas chromatography/mass spectrometry but was easily measured by gas chromatography/isotope ratio mass spectrometry. These assays should be useful for clinical investigations, in which the incorporation of a (13C)gluconeogenic substrate into glucose must be corrected for the incorporation of 13CO2 derived from the oxidation of the substrate. This correction involves measuring the low-level 13C enrichment of urea.