Xenoestrogens are defined as chemicals that mimic some structural parts of the physiological estrogen compounds, therefore may act as estrogens or could interfere with the actions of endogenous estrogens. Two subtypes of the ER are known, the ER alpha and ER beta, and both have a distinct tissue distribution and play a distinct role in physiology. Receptor dimmer assumes a distinctive conformation, binds to its estrogen response element (ERE), interacts with the general transcription complex bound to the TATA box within the respective gene promoter, and regulates gene transcription. The discovery and identification of co-activators and co-repressors provided crucial insights into the ER action. New evidence indicates that the activation of additional transcription factors as well as the action of xenoestrogens through estrogen receptors located outside the cell nucleus (in the plasma membrane, mitochondria and probably the cytosol) should be considered. The levels of exposition to xenoestrogens and the age of the investigated animal can have a significant effect on its development and reproduction. Therefore, several in vivo and in vitro assays have been developed to assess the estrogenic-like activity of individual compounds or natural mixtures. In this review, selected methods applied in physiological studies have been described. One of the most extensively used in vivo assays for estrogenicity is the rodent uterotrophic assay. In order to analyze the estrogenic properties of xenoestrogens, morphological, histological, biochemical and molecular studies should be introduced. A variety of in vitro tests have been established to determine estrogenic potency of xenoestrogens but even a combination of them is not able to predict their actual action in the organism. There is a need for the studies on all potential xenoestrogens to describe tissue-specific activities, and via which pathways in those tissues these compounds either disrupt or mimic hormone action.