PHI-443 (N′-[2-(2-thiophene)ethyl]-N′-[2-(5-bromopyridyl)] thiourea) is a rationally designed novel thiophene thiourea nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent anti-HIV activity against the wild-type and drug-resistant primary clinical human immunodeficiency virus (HIV-1) isolates. This study examined the potential utility of PHI-443 as a nonspermicidal microbicide for prevention of sexual transmission of HIV. Our goal in this study was to test the effects of PHI-443 on in vivo sperm functions under conditions shown to inactivate viruses in human cells. PHI-443 completely prevented the vaginal transmission of a genotypically and phenotypically drug-resistant HIV-1 isolate in the humanized severe combined immunodeficient (Hu-SCID) mouse model of sexually transmitted AIDS. Exposure of human sperm to PHI-443 at doses 30 000 times higher than those that yield effective concentrations against the AIDS virus had no adverse effect on sperm motility, kinematics, cervical mucus penetrability, or the viability of vaginal and cervical epithelial cells. Exposure of rabbit semen to PHI-443 either ex vivo or in vivo had no adverse impact on in vivo fertilizing ability in the rabbit model. Reproductive indices (i.e., pregnancy rate, embryo implantation, and preimplantation losses) were not affected by pretreatment of rabbit semen with PHI-443. Likewise, intravaginal application of 2% PHI-443 via a self-emulsifying gel at the time of artificial insemination resulted in healthy offspring with no apparent peri- or postnatal repercussions. Repeated intravaginal administration of 0.5%– 2% PHI-443 gel was found to be safe in rabbits and lacked systemic absorption. PHI-443 has clinical potential as a prophylactic broad-spectrum anti-HIV microbicide without contraceptive activity.