To investigate the role of telomerase in estrogen-regulated rodent tissues, we assayed the activity levels of this enzyme and measured cell proliferation and indicators of cellularity in vagina, mammary gland, and uterus from virgin, pregnant, ovariectomized, and ovariectomized estradiol-treated rats. No association was observed between telomerase activity and increased cell proliferation. Telomerase activity was significantly higher (P=0.003) in vagina obtained from ovariectomized rats (very low proliferation) than in vagina from ovariectomized and estradiol-treated rats (high proliferation, high differentiation). The high telomerase levels observed in vagina from ovariectomized rats indicates that the same epithelial compartment (i.e., basal layer) that has the potential to reconstitute the epithelium also contains the cells that express telomerase. The lower telomerase activity in the keratinized (differentiated) vagina was probably due to dilution of the number of telomerase-producing cells by the terminally differentiated non-telomerase-producing cells. Similar results were observed in uterus from ovariectomized versus ovariectomized and estradiol-treated rats. Telomerase activity was highest in uterus from pregnant rats. Telomerase levels in samples from total mammary gland fat pads varied considerably between groups and appeared to be representative of the amount of epithelium present in the sample. Interestingly, when mammary gland samples from the same animals were obtained from pure epithelial organoid preparations, no differences in telomerase activity could be distinguished between animals or groups. Overall these data suggest that telomerase activity, particularly in rat vagina and uterus, appears to be associated with a cell subpopulation showing proliferative and tissue reconstitution potential and not directly associated with proliferation status per se.