The relative effect of HIV-1 infection compared with vaginal infections on vaginal cytokine concentrations is not well characterized. We compared vaginal fluid samples from HIV-1-infected women with those from HIV-negative women, to assess the effect of HIV-1 infection on concentrations of vaginal proinflammatory cytokines and the mucosal defense molecule secretory leukocyte protease inhibitor (SLPI). Twenty-seven HIV-1-infected women and 54 HIV-negative controls, matched for bacterial vaginosis (BV) status, had proinflammatory cytokine [interleukin (IL)-1beta, IL-6, IL-8] and SLPI concentrations measured from archived cervicovaginal lavage and vaginal swab samples using an enzyme-linked immunosorbent assay (ELISA). Log-transformed concentrations were compared by BV and HIV status in univariate analysis using Student's t-test, and in multivariate analysis using a linear regression model. In univariate analysis there were no significant differences in cytokine concentrations among HIV-1-infected and HIV-negative women. In a multivariable linear regression model, BV was significantly associated with an increase in IL-1 beta (p = 0.003). HIV infection was associated with an increased concentration of SLPI (p = 0.008), while BV status was significantly associated with a decrease in SLPI concentrations (p = 0.005). Neither HIV nor BV was associated with changes in IL-6 or IL-8. HIV does not have a major impact on vaginal concentrations of proinflammatory cytokines when controlling for the presence of bacterial vaginosis.