The rising prevalence of human immunodeficiency virus type 1 (HIV-1) infection in women, especially in resource-limited settings, accentuates the need for accessible, inexpensive, and female-controlled preexposure prophylaxis strategies to prevent mucosal transmission of the virus. While many compounds can inactivate HIV-1 in vitro, evaluation in animal models for mucosal transmission of virus may help identify which approaches will be effective in vivo. Macaques challenged intravaginally with pathogenic simian immunodeficiency virus (SIV(mac251)) provide a model to preclinically evaluate candidate microbicides. 2-Hydroxypropyl-beta-cyclodextrin (BCD) prevents HIV-1 and SIV infection of target cells at subtoxic doses in vitro. Consistent with these findings, intravaginal challenge of macaques with SIV(mac251) preincubated with BCD prevented mucosal transmission, as measured by plasma viremia and antiviral antibodies, through 10 weeks postchallenge. In an initial challenge, BCD applied topically prior to SIV(mac251) prevented intravaginal transmission of virus compared to controls (P < 0.0001). However, upon a second virus challenge following BCD pretreatment, the majority of the previously protected animals became infected. The mechanism through which animals become infected at a frequency similar to that of controls after prior exposure to BCD and SIV(mac251) in subsequent intravaginal virus challenges (P = 0.63), despite the potent antiviral properties of BCD, remains to be determined. These results highlight the unpredictability of antiviral compounds as topical microbicides and suggest that repeated exposures to candidate treatments should be considered for in vivo evaluation.