Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERα and ERβ. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERβ expressed in rat tissues, β1 and β2, may function as inhibitors of ERα transcriptional activity. To gain insight into the role of ERβ in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERβ1 and ERβ2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17β-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERβ1 and ERβ2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERα mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERβs in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERβ expression may be required for estrogen to exert its full trophic effects on the uterus.