A rise in new HIV diagnoses among older adults is characterized by poor prognosis and reduced survival times. Although heterosexual transmission remains the main route of infection in women, little is known regarding immune functions in the genital tract of postmenopausal women, especially those who are HIV positive. Furthermore, effects of hormone replacement therapy (HRT) on the genital tract immune system are unclear. Using the Women's Interagency HIV Study repository, we obtained cervical-vaginal lavage (CVL) samples from premenopausal and postmenopausal HIV-positive and HIV-negative women, some of whom were on HRT. Samples were assayed for interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-a, secretory leukocyte protease inhibitor (SLPI), Elafin, human beta defensin-2 (HBD2), and macrophage inflammatory protein (MIP)-3a using ELISA. Anti-HIV activity in CVL was measured using TZM-bl indicator cells. Among HIV-positive women, the plasma viral load was significantly higher and CD4 count was significantly lower in postmenopausal compared with premenopausal women. Postmenopausal women, irrespective of HIV status, had significantly lower levels of HBD2 compared with premenopausal women. Among the HIV-negative individuals, postmenopausal women had significantly lower levels of MIP-3a, IL-6, and SLPI compared with premenopausal women. In contrast, HIV-positive postmenopausal women had significantly higher levels of TNF-a compared with HIV-positive premenopausal women. In most cases, HRT groups resembled the postmenopausal groups. No significant differences in anti-HIV activity by menopausal or by HIV status were noted. Our findings indicate that the female genital tract immune microenvironment is distinct by menopausal status and HIV status. Further studies are needed to assess the risk of HIV acquisition/transmission in this population.