beta-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production and innate defences are crucial to the preservation of fertility and successful pregnancy. This study details the expression of the recently characterized defensins, HBD3 and 4, in human endometrium. Using real-time quantitative RT-PCR, we have shown that HBD3 mRNA expression is highest during the secretory phase of the menstrual cycle while HBD4 mRNA levels peak in the proliferative phase. Both antimicrobials are expressed by endometrial epithelium. Exogenous steroid hormones in the form of the combined oral contraceptive pill (COCP) alter expression of both defensins in vivo, while treatment of endometrial explants with progesterone in vitro does not alter expression of HBD3 or HBD4. In in vitro cultures of primary endometrial epithelial cells, HBD3 mRNA expression is upregulated by treatment with inflammatory molecules including IL-1 beta+TNF alpha, IFN gamma and phorbol ester. HBD4 mRNA was not expressed in these primary cell cultures. These results show that the human endometrium expresses both HBD3 and HBD4 in a cycle-dependent manner. These natural antimicrobials will contribute to innate defences present in human endometrium protecting against uterine infection. Expression is altered as a result of hormonal contraceptive use and this may contribute to differential infection rates in COCP users relative to non-users. In addition, expression of HBD3 will be upregulated during infection allowing an increased innate immune response at this time.