Endometriosis and uterine fibroids (leiomyomas) are benign gynecological disorders affecting 5-15% of women of reproductive age. They cause a wide range of symptoms including mild to severe pelvic pain and infertility. Due to a paucity of proper study models, hormonal and cellular mechanisms driving the pathology of endometriosis and fibroid development and growth remain unclear. Therefore, in the current study, we established 3D spheroid/organoid cultures from human uterine epithelial and Stro-1+/CD44+ myometrial stem cells and also from cells isolated from normal proliferative phase endometrium. Uterine organoid cultures were derived from endometrial epithelial and myometrial cells isolated from women who were not receiving exogenous hormones at the time of laparoscopy or hysterectomy. They were embedded in Matrigel, and grown in culture media. To determine whether spheroids/organoids were responsive to steroid hormones, the cultures were treated in presence or absence of estradiol (E2), progesterone (P4) or the combination (E2+P4) in serum free culture media. Time-dependent spheroid/organoid-growth curves and morphological analyses were used to define growth characteristics of endometrial and myometrial organoids. Subsequently, immunohistochemical colocalization of steroid hormone receptors (estrogen receptor alpha (ER-α) and progesterone receptor (PR-A\\B), alpha smooth muscle actin (α-SMA; myometrial cell marker), vimentin (stromal cell marker) and E-cadherin (endometrial epithelial cell marker) was assessed. Epithelial organoids expressed only E-cadherin in the absence of hormonal treatment. Myometrial organoids expressed α-SMA and vimentin. No expression of E-cadherin was observed in myometrial organoids. However, we observed the expression of ER-α and PR-A\\B when organoids were treated with E2+P4 in a time-dependent manner. Stro-1+/CD44+ myometrial stem cells differentiated into α-SMA and fibroblast/stromal cells and response to sex hormones. These findings suggest human uterine organoid cultures retained their characteristic cellular responses to E2+P4 and could be maintained long-term in ex vivo culture. Thus, the current 3D uterine organoid systems show high expansion capacity with retention of phenotypical and functional properties, which can be used for uterine pathophysiological studies, drug discoveries and drug repositioning.