The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.