The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS-1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β-estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS-4, -5, -8 or -9 expression in human endometrial stromal cells in vitro. Real-time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS-8, -9 and -5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4- or DHT-mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization.