Humans are exposed to phthalates due to the ubiquitous use of these chemicals in consumer products. In the body, phthalates metabolize quickly to form hydrolytic and oxidative monoesters which, in turn, can be glucuronidated before urinary excretion. Exposure assessment studies typically report the total urinary concentrations of phthalate metabolites (i.e., free plus glucuronidated species). Nevertheless, because conjugation may potentially reduce the bioactivity of the metabolites by reducing their bioavailability, measuring the concentrations of free species may be of interest. An accurate, quantitative measurement of phthalate monoesters and their conjugated species requires data on the stability of these species in urine after sample collection and before analysis. We studied the stability of eight phthalate metabolites and their glucuronide conjugates at 25, 4, and -70 degrees C. Interestingly, the total concentrations of phthalate metabolites decreased over time at 25 and 4 degrees C, but not at -70 degrees C for up to 1 year and despite several freeze-thaw cycles. We further observed a considerable decrease in the concentrations of the glucuronides of some phthalate metabolites 1 day and 3 days after collection when the samples were stored at 25 and 4 degrees C, respectively. By contrast, the concentrations of the glucuronide conjugates at -70 degrees C remained unchanged for the whole duration of the study (1 year). Based on these findings, we recommend transferring urine specimens to a cooler or a refrigerator immediately after collection followed by permanent storage at subfreezing temperatures within hours of sample collection.