Dietary isoflavones are biologically active in humans, but few observational data exist on the relationship between isoflavone intake and excretion in Western populations. We examined associations between self-reported soy intakes and overnight urinary isoflavone excretion in a population-based sample of western Washington State women, and we investigated the usefulness of one versus two overnight urine samples, collected 48 h apart, as a biomarker of intake. Isoflavones (genistein, daidzein, O-desmethylangolensin, and equol) were measured in two overnight urine collections from 363 women recruited from a health maintenance organization. Soy food intakes were assessed using two 1-day diet records completed on each day prior to the urine collections and a food frequency questionnaire (FFQ) that had been completed by 312 of the women with regard to their dietary habits 3.5 years (range, 2-5 years) before the urine collections. Twenty-one percent of the women consumed soy on either day of the diet recall, and 13% and 34% of the women consumed soy at least once a week or at least once a month, respectively, according to the FFQ. Women who consumed soy at either of the two diet recalls or at the FFQ (at least once a week or at least once a month) had a significantly higher urinary excretion of isoflavones than women who did not consume soy (P < 0.01). Among women who consumed soy at either of the two diet recalls or at the FFQ (soy consumed at least once a month), isoflavone intake and excretion correlated significantly (P < 0.01). Excretion of the individual isoflavones correlated significantly between the two urine samples collected 48 h apart (genistein, r = 0.41 and P < 0.001; daidzein, r = 0.30 and P < 0.001; O-desmethylangolensin, r = 0.46 and P < 0.001; equol, r = 0.60 and P < 0.001). Differences between soy consumers and nonconsumers and associations between intakes and excretion remained significant whether one or both urine collections were considered. Measuring isoflavone excretion in one overnight urine collection serves as a biomarker of recent or past isoflavone intake, even in populations whose intake of soy foods is relatively low.