The phytoestrogen enterolactone has been hypothesized to prevent breast cancer. Because one of the biological effects of enterolactone is probably estrogenic, it is possible that the preventive effect on breast cancer differs with the estrogen receptor (ER) alpha status of the tumor. The objective of this study was to investigate whether high plasma levels of enterolactone are associated with breast cancer risk and whether the ERalpha status of the tumor influences this relation. The cohort study Diet, Cancer and Health included 29,785 women, ages 50 to 64 years, between 1993 and 1997. Information about diet and life-style factors was obtained by questionnaire, and blood was drawn from each participant. We matched 381 postmenopausal breast cancer cases to 381 controls and analyzed the concentration of enterolactone in plasma with a time-resolved fluoroimmunoassay. Associations between plasma concentrations of enterolactone and breast cancer were analyzed by logistic regression. The incidence rate ratio (IRR) for all breast cancer was 0.93 [95% confidence interval (CI), 0.86-1.01] per 20 nmol/L higher plasma concentration of enterolactone. For ERalpha-positive cancers (n=273) only a weak association was seen (IRR, 0.97; 95% CI, 0.88-1.06), whereas for ERalpha-negative cancers (n = 80; IRR, 0.71; 95% CI, 0.53-0.94) a protective effect was seen per 20 nmol/L higher plasma enterolactone. In accordance with earlier research, we found a tendency toward a lower risk for breast cancer with higher concentrations of enterolactone, which was restricted almost entirely to ERalpha-negative breast cancer.