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)-kappaB pathway. We investigated the effects of E2 treatment and adenovirus-mediated estrogen receptor (ER)-alpha gene transfer on cell proliferation and NF-kappaB 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 E(2) on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER-alpha gene into VSM cells. Constitutive and interleukin (IL)-1beta-stimulated NF-kappaB 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-1beta-stimulated NF-kappaB activation. ER-alpha 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-1beta-stimulated NF-kappaB 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-alpha gene transfer and E2 treatment. In addition, increased NF-kappaB signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.