A thermospray liquid chromatographic/mass spectrometric method has been developed for direct determination of cotinine-N-glucuronide in the urine of smokers. Quantification was performed using methyl-d3-cotinine-N-glucuronide as internal standard and monitoring the protonated aglycons. Using a simple preparation, urine samples from four smokers were analyzed and the results compared favorably with those from a previously reported method that quantifies aglycon release following beta-glucuronidase treatment. Amounts of cotinine-N-glucuronide found in urine from smokers ranged from less than 0.7 to 21 nmol ml-1, indicating wide inter-individual variability in the metabolic production of this metabolite. Cotinine-N-glucuronide was found to be the second most abundant urinary nicotine metabolite. A similar method was developed for trans-3'-hydroxycotinine-N-glucuronide but this compound was not detected in smokers' urine.