The presence of cotinine, a nicotine metabolite, in urine above a specified cutoff concentration is commonly used to distinguish smokers from nonsmokers, as in smoking cessation studies. A stability study of cotinine in urine was carried out after questions arose concerning analyte stability at elevated storage and shipment temperatures. Aliquots from a smokers urine pool were stored at 5, 25, 40, 50 and 60 degrees C for 30 days. Another set of aliquots, obtained by diluting the smokers pool 1:1 with nonsmokers urine, were stored under the same conditions. Free cotinine levels, determined by a stability-indicating gas chromatographic/mass spectrometric (GC/MS) assay, increased over the 30-day period at higher storage temperatures. Cotinine concentrations in the aliquots stored at 60 degrees C, for example, nearly doubled over 30 days (1301 to 2476 ng/ml), with similar proportional increases observed in the aliquots diluted with nonsmokers urine. Since cotinine can be excreted to a large degree as cotinine-N-glucuronide, this conjugated metabolite was determined by an indirect method. As the storage temperature increased, the free/conjugated cotinine ratio dramatically increased, pointing to cotinine-N-glucuronide as the source of the additional free cotinine at the higher storage temperatures. The results of this study are of much practical importance, since urine samples with free cotinine concentrations just below a cutoff value may become positive for smoking status if suitably low temperatures cannot be maintained during sample handling and shipment.