Objective To determine the vaginal contraceptive activity of WHI-07 in the rabbit model. Design Prospective, controlled study. Setting Center for advanced preclinical sciences. Animal(s) Subgroups of 15, 16, or 24 New Zealand White does and 24 bucks per experiment. Intervention(s) Ex vivo (Experiment 1) and in vivo (Experiments 2 and 3) treatment of semen with WHI-07 or Nonoxynol-9 (N-9). In Experiment I, ovulated does in subgroups of 15 were artificially inseminated with semen mixed with WHI-07 or vehicle. In Experiment 2, ovulated does in subgroups of 24 were artificially inseminated within 2 min after intravaginal administration of 2% WHI-07 gel-microemulsion or 2% N-9 gel and allowed to complete term pregnancy. In Experiment 3, ovulated does in subgroups of 16 were artificially inseminated at 15, 30, or 60 minutes. Main outcome measure(s) The numbers of implanted embryos on postinsemination day 8 or the proportion of does that became pregnant and delivered newborn rabbits; the litter size, weight, growth, and viability of pups until lactation day 5. Result(s) Exposure of semen to WHI-07 at the time of artificial insemination completely inhibited pregnancy rates (WHI-07–pretreated, 0%, vs. control, 60%) and embryo implantation (WHI-07-pretreated, 0/175 vs. control, 68/170). Intravaginal administration of a 2% WHI-07 gel-microemulsion or 2% N-9 gel before artificial insemination significantly inhibited pregnancy rates (81% and 85% inhibition, respectively) when compared with control. Furthermore, the 2% WHI-07 gel-microemulsion provided ⪢90% inhibition of fertility even when insemination was delayed until 60 minutes after intravaginal application. Rabbits that delivered litters despite intravaginal application of 2% WHI-07 gel-microemulsion had healthy offsprings with no perinatal or postnatal repercussions. Conclusion(s) WHI-07 is a potent contraceptive agent in vivo. Intravaginal use of WHI-07 gel-microemulsion has clinical potential as a safe prophylactic contraceptive, in addition to its microbicide activity to curb the sexual transmission of HIV.