The ratio of urinary 6beta-hydroxycortisol:cortisol is a measure of the activity of cytochrome p450 3A4 (CYP3A4). CYP3A4 catalyzes the formation of the genotoxic estrogen, 16alpha-hydroxyestrone. It is also involved in the activation of many other mammary carcinogens, such as the polycyclic aromatic hydrocarbons and heterocyclic amines. We evaluated the association between urinary cortisol ratios and breast cancer risk in a subgroup of women who participated in a population-based case-control study in Shanghai. Overnight urine samples from 246 case-control pairs were assayed for 6beta-hydroxycortisol (6beta-OHC) to cortisol. The urine samples from all of the breast cancer patients were collected before any chemotherapy or radiotherapy. In-person interviews were conducted to obtain comprehensive information on dietary habits, reproductive history, and other lifestyle factors. The median levels of 6beta-OHC:cortisol ratios were 2.61 in cases and 2.16 in controls, a 20.8% difference (P < 0.001). The case-control difference was larger in women over 45 years of age (31.3% difference; P < 0.001) than younger women (6.0%; P = 0.45). After adjusting for confounding variables, the risks of breast cancer were increased from 1.0 (reference) to 1.6 [95% confidence interval (CI), 0.9-3.1], 2.2 (95% CI, 1.1-4.2), and 3.7 (95% CI, 1.9-7.4; P for trend, <0.001) with increasing levels of 6beta-OHC:cortisol ratios. The positive association was more pronounced among older women (>45 years) than among younger women (< or = 45 years). The adjusted odds ratios associated with the highest cortisol ratio were 6.0 (95%CI, 2.2-16.1) among older women and 2.2 (95%CI, 0.8-6.1) among younger women. The association of the 6beta-OHC:cortisol ratio was stronger among older women who had a high body mass index, late age at menopause, and early age at menarche (factors related to high endogenous estrogen exposure) than those who did not have these factors. These findings are consistent with the role of CYP3A4 in estrogen and carcinogen metabolism and suggest that high CYP3A4 activity may be a risk factor for breast cancer risk.