Gas chromatography-high resolution mass spectrometry methods were developed for quantifying nitrosodimethylamine (NDMA) and nitrosoproline (NPRO) in human urine. The limits of quantitation of these methods, which utilize stable isotope analogues of NDMA and NPRO as internal standards, were 5 pg per ml for NDMA and 0.14 ng per ml for NPRO. The assays were used to quantify NDMA and NPRO in urine samples collected 4 times a wk for 5 wk from 24 healthy volunteers. The mean urinary excretion of NDMA during this time was found to be 38.2 ng per day, and the mean urinary excretion of NPRO was found to be 3.26 µg per day. Treatment of the volunteers with 600 mg of ascorbic acid and 100 IU of α-tocopherol 4 times a day for the final 3 wk of the study did not influence the urinary excretion of NDMA or NPRO. Considerable person-to-person and day-to-day variations were observed for the urinary excretion of both nitrosamines, but the urinary excretion of NDMA was not correlated with the urinary excretion of NPRO. Person-to-person and day-to-day differences in the urinary excretion were greater for NPRO than for NDMA. The mean urinary excretion of NDMA by the 24 subjects was as much as 5-fold higher on some days than on other days, but this was not observed for NPRO. Day-to-day differences in the mean urinary excretion of NDMA were correlated with the concentrations of nitrogen dioxide in the air.