Female ROP Os/+ mice are partially protected by endogenous estrogens against progressive glomerulosclerosis (GS) during their reproductive period; however, ovariectomy accelerates GS progression. We examined the effects of continuous and intermittent 17beta-estradiol (E(2)) replacement and tamoxifen therapy on the development of GS in ovariectomized (Ovx) ROP Os/+ mice. Continuous E(2) replacement (CE(2)) throughout 9 months prevented microalbuminuria and excess extracellular matrix accumulation in Ovx ROP Os/+, not only compared to placebo-treated Ovx mice but also in comparison to intact female ROP Os/+. Tamoxifen had a similar effect, but of lesser magnitude. Intermittent 3-month on-off-on E(2) did not reduce the kidney changes. Mesangial cells (MCs) from CE(2) mice maintained their estrogen responsiveness. E(2) in vitro prevented transforming growth factor-beta1 stimulation of a Smad-responsive reporter construct and increased MMP-2 expression and activity in MCs isolated from CE(2) mice. MCs from mice on either placebo or intermittent E(2) treatment did not respond to added E(2), consistent with a stable alteration of their estrogen responsiveness. Tamoxifen protection against GS was less pronounced in ROP Os/+ mice. Thus, prolonged estrogen deficiency promotes GS and renders MCs insensitive to subsequent estrogen treatment. This underscores the importance of continuous estrogen exposure for maintaining glomerular function and structure in females susceptible to progressive GS.