Epidemiological studies have demonstrated that hormone replacement therapy with estrogen (E₂) or E₂ plus progesterone in postmenopausal women decreases the age-associated risk of cardiovascular disease by 30-50%. Treatment of vascular smooth muscle (VSM) cells with physiological concentrations of E₂ has been shown to inhibit growth factor-stimulated cell proliferation. In this study, we tested the hypothesis that E₂ inhibits the age-associated increase in VSM cell proliferation by inhibiting nuclear factor (NF)-B pathway. We investigated the effects of E₂ treatment and adenovirus-mediated estrogen receptor (ER)- gene transfer on cell proliferation and NF-B activation using VSM cells cultured from 3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstrate that VSM cell proliferation was significantly increased (P < 0.05) in aged compared with young adult female rats. Treatment of VSM cells with physiological concentrations of E₂ inhibited VSM cell proliferation, and this inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects of E₂ on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER- gene into VSM cells. Constitutive and interleukin (IL)-1-stimulated NF-B activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats. E₂ treatment of VSM cells from aged female rats inhibited both constitutive and IL-1-stimulated NF-B activation. ER- gene transfer into VSM cells from aged female rats further augmented the inhibitory effects of E₂. In conclusion, our data demonstrate that constitutive and IL-1-stimulated NF-B activation is increased in VSM cells from aged female rats due to loss of E₂ and this can be restored back to normal levels by ER- gene transfer and E₂ treatment. In addition, increased NF-B signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.