The Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) estimates that as many as 80,000 endocrine disruptor compounds (EDCs) should be evaluated to determine their estrogenic or anti-estrogenic activity. The development of standardized protocols to test for hormonally active EDCs is a major goal of regulatory agencies worldwide. Our studies focus on the potential for EDCs being present in the micro-environment of the research animals used for EDC studies (e.g., diet, caging, bedding, and environmental enrichment) and the impact these compounds may have on study outcomes, especially reproductive development. In the past, we have utilized either changes in uterine weight or the timing of vaginal opening (VO) in rodents, both estrogen-sensitive endpoints. Currently, we have focused on the effects on VO, as we feel this is a sensitive indicator of reproductive development. In our studies, pre-pubertal CD-1 mice are weaned at post-natal day (PND) 15 and given test compounds orally (via diet, water or gavage) from PND 15 to PND 35 or until VO occur to determine the effects of on the timing of VO. Vaginal opening is recorded daily from weaning to time of VO. We have shown that natural phytoestrogens (e.g., daidzein and genistein) present in commercially available rodent diets or added to diets free of these compounds can significantly (P less than 0.01) accelerate the time of VO in CD-1 mice. We have also shown that the total metabolizable energy (ME) in the diet can significantly affect these hormone-sensitive endpoints, although the predictability of this variable was less powerful than the phytoestrogen content. We have shown that the estrogenic mycotoxin zearalenone is ubiquitious in commercially available corn-cob bedding. 154 of 189 (84%) of the samples were naturally contaminated with zearalenone at levels ranging from 100 to 7,000 ppb (mean 500 ppb). We have shown that levels of 5-10 ppm can significantly advance the time of VO in immature CD-1 haired and SKH-1 hairless mice. Bisphenol A (BPA) is a highly publicized EDC compound. We have been concerned that animal studies on the ED effects of BPA or other EDCs have not adequately or consistently controlled for the potential EDC effects of the animals micro-environment that could affect the study outcome. The use of high phytoestrogen diets or diets with high or variable levels of ME or the use of corncob bedding can dramatically alter EDC studies. We have also had concerns that the route of BPA exposure in some reports may not adequately assess risks to human exposures. We have found that reports on low dose BPA (50 g /kg bwt/day) animal studies are inconsistent, and the use of high phytoestrogen diets and the subcutaneous route of exposure are contributing factors. Our literature review further indicates that the identification and reporting of the diet, bedding, caging and water bottles used in BPA studies are not consistent. Currently, we are determining the effects of BPA and other estrogenic monomers leaching from the various types of animal cages, environmental enrichment and water bottles and their impact on the time of VO in immature CD-1 mice.