Urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronide, which are metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are valuable biomarkers of tobacco exposure. In epidemiologic investigations, it is often necessary for samples to be collected and stored for various periods of time prior to analysis, and as a consequence, it is important to determine the stability of these analytes during storage. In this study, two urine pools were stored at six different temperatures ranging from -70 degrees C to 55 degrees C. Free NNAL and NNAL-glucuronide were measured at scheduled time intervals for 28 days, after which samples stored at -20 degrees C and -70 degrees C were further monitored for an additional four years. NNAL-glucuronide was not completely stable at room temperature (ca. 22 degrees C) or above. At those temperatures, a gradual loss of NNAL-glucuronide with concomitant increases in free NNAL was observed. However, both free NNAL and NNAL-glucuronide appeared to be stable for at least four weeks when stored in the refrigerator (4 degrees C) and for at least four years at -20 degrees C and -70 degrees C, respectively. These results suggest that valid NNAL concentrations can be measured in frozen urine samples that have been stored for an extended period of time prior to analysis.