In rat endometrium, expression of gap junction connexin-26 (cx26) in the epithelium and cx43 in the uterine stroma is suppressed by progesterone before implantation. For further study of connexin gene regulation we analyzed expression of cx26, cx43, and cx32 in the endometrium of ovariectomized rats treated with different ratios of 17beta-estradiol (E2) and progesterone (P). A hormonal ratio of E2 to P that mimics conditions during pregnancy (0.1 microg E2 and 4 mg P) suppressed expression of cx26 and cx43. By changing the ratio to higher E2 levels (1 microg E2), cx26, in contrast to cx43, was not suppressed even by application of a high P concentration (10 mg). Time-course experiments supplying E2 alone led to an early gene response of cx26 within 3 h, whereas induction of cx43 transcripts was not detected until 14 h after E2 treatment. Simultaneous application of the antiestrogen ICI 182780 abolished E2-mediated induction of both connexins. No hormonal regulation of cx32 could be detected. As already shown for cx43 gene induction in the myometrium, E2-mediated induction of cx26 expression in the endometrium also required newly synthesized transcription factors. It can be concluded that only a hormonal ratio resembling conditions during pregnancy is able to suppress the expression of both cx26 and cx43 and that cx26 gene expression is induced earlier by E2 and is likely to be more sensitive to a shift in the E2 to P ratio than cx43.