17-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17- estradiol is locally synthesized in non-reproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes and Peyer’s patches are novel sites of de novo synthesis of 17-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein co-expressed in cells that express aromatase, the enzyme responsible for 17-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed aromatase expression was localized to high endothelial venules (HEVs) of these lymphoid organs, and these HEV cells synthesized 17-estradiol when isolated and cultured in vitro. Both the mesenteric lymph nodes and Peyer’s patches contained 17-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor beta (ER) had hypertrophic Peyer’s patches and mesenteric lymph nodes withmoreleukocytes than their wild type littermates, demonstrating a role for 17-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17-estradiol content, or steroidogenic capacity in these lymphoid organs.