Two studies were conducted to assess factors affecting estrogen excretion in dairy heifers. The objective of the first study was to quantify estrogenic activity in feces and urine during the estrous cycle. Ten non-pregnant Holstein heifers were fed the same diet for 28 d. Plasma, feces, and urine samples were collected daily. Plasma 17-β estradiol (17-β E2) was quantified with RIA and used to confirm day of estrous. Feces and urine samples from days -12, -6, -2, -1, 0, 1, 2, 6, 12 of the estrous cycle were analyzed with RIA and Yeast Estrogen Screen (YES) bioassay. Plasma 17-β E2 concentrations peaked on day of estrus, with feces and urine estrogenic excretion peaking a day after. The objective of the second study was to quantify variation in estrogenic activity in feces and urine due to increased dietary phytoestrogen content. Ten Holstein heifers were randomly assigned to treatment sequence in a two-period crossover design. Dietary treatments consisted of grass or red clover hay, and necessary supplements. Feces and urine samples were collected and pooled for analysis. Estrogenic activities of pooled samples were quantified using the YES bioassay. Estrogenic excretion in feces and urine was higher for heifers fed red clover hay. Fecal and urine samples from five heifers were analyzed using LC/MS/MS to quantify excretion of phytoestrogenic compounds. Heifers fed red clover hay excreted more equol than heifers fed grass. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation.