Estrogens are primarily synthesized in the gonads both in males and females and regulate the development and function of reproductive organs, undoubtedly as gonadal sex hormones. Recent studies however find that estrogens also play important roles in regulating normal and patho-biological processes in non-reproductive organs that are critical for health in humans and animals alike. Furthermore, recent findings show that estrogens are also synthesized in extra-gonadal sites and play an equally important role in non-reproductive systems. These exciting discoveries highlight a great potential for the use of estrogen as a therapeutic target in disease prevention and treatment. Yet developing a targeting strategy remains challenging because knowledge of extra-gonadal estrogen biosynthesis, and the mechanisms by which estrogens act are very limited. In a previous study, we discovered that 17?-estradiol (E2) is synthesized de novo in the intestinal lymphoid tissues in mice and plays a role in regulating leukocyte homeostasis. This finding led us to hypothesize that E2 is synthesized in the intestinal lymphoid tissues across the entire mammalian species. In this study, we investigate procine E2 synthesis in the intestinal lymphoid tissues. Porcine species are similar to humans in genetic, physiological, anatomical and developmental aspects of gastrointestinal tract and immune systems, making them an excellent model system for medical research on human. My study found that cultured mesenteric lymph node (mLN) and Peyer’s patch (Pp) synthesized estradiol whereas ileum did not. mLN showed and Pp had 60% and 30% estradiol synthetic capacity compared to that of ovary, respectively. Addition of androstenedione, precursor of estradiol, further increased E2 synthesis. Messenger RNAs for steriodogenic enzymes (StAR, Hsd17b, Hsd3b, Cyp17, Cyp19) were found in both mLN and Pp, comparable levels to the those in the ovary. In the mLN, aromatase (Cyp19) protein was expressed the endothelial cells of high endothelial venule (HEV). Taken together, HEV endothelial cells in intestinal lymphoid tissues of porcine GI tract synthesize E2 de novo.