Objective: We undertook a prospective study to assess the impact of HIV infection on BMD in a cohort of HIV-infected and uninfected women that included illicit drug users, and to measure the contribution of traditional risk factors as well as HIV-related factors to loss of BMD over time. Methods: We analyzed BMD at baseline and after ≥18 months in 245 middle-aged HIV-infected and 219 uninfected women, and conducted linear regression analysis to determine factors associated with annual BMD change at the femoral neck, total hip and lumbar spine. Results: HIV-infected women had lower baseline BMD at the femoral neck and total hip compared with controls; unadjusted rates of BMD change did not differ by HIV status at any site. In multivariable analyses, we found that HIV seropositivity without protease inhibitor (PI) use was associated with BMD decline at the lumbar spine (-.009 g/cm2 per year, p =.03). Additional factors associated with BMD decline were: postmenopausal status, lower BMI, and methadone use at the lumbar spine; postmenopausal status and hepatitis C seropositivity at the femoral neck; and postmenopausal status, age, smoking, and lower BMI at the total hip (all p <.05). Among HIV-infected women, ≥3 years of PI use was associated with an increase in lumbar spine BMD (.013 g/cm2 per year, p =.008). Conclusions: Bone loss among HIV-infected middle-aged women was modest, and possibly mitigated by PI use. Methadone use was associated with BMD decline, and should be considered when evaluating women for osteoporosis risk.