BACKGROUND Epigenetic regulation may be involved in modulation of gene expression during the normal cyclic changes of the human endometrium. We investigated expression of DNA methyltransferases (DNMTs) in endometrium during the menstrual cycle and the influence of sex steroid hormones on DNMT in endometrial stromal cells (ESC) in culture. METHODS Expression of DNMT1, DNMT3a and DNMT3b was assessed by immunohistochemistry and real-time RT–PCR in endometrial tissue (n = 42 women). ESC (n = 3 women) were cultured with estradiol and medroxyprogesterone acetate (E + MPA) for 17 days, and DNMT mRNA levels were measured by real-time RT–PCR. RESULTS Nuclei of both epithelial and stromal cells immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Tissue levels of DNMT1 and DNMT3a mRNA were significantly lower in the mid-secretory phase than in the proliferative phase (P < 0.01). For DNMT3b, the change in mRNA levels showed a similar trend to that for DNMT3a. In ESC culture, DNMT3a and DNMT3b mRNA levels were significantly decreased by E + MPA treatment (P < 0.01 and P < 0.05, respectively) at Day 8 and Day 17. CONCLUSIONS DNMT mRNAs declined in the human endometrium during the secretory phase, and E + MPA down-regulated DNMT3a and DNMT3b mRNAs in ESC in culture. These results suggest that DNMTs have regulatory functions in gene expression that is associated with decidualization.