Pretreating the rectum of mice with nonoxynol-9 (N9) or spermicides containing N9 before infecting via the rectal route with herpes simplex virus 2 (HSV-2) increased the likelihood of infection and shortened the time until infection. Microscopic analysis indicated that N9 rapidly caused exfoliation of the rectal epithelium, exposing the underlying connective tissue. These findings suggest that use of N9-containing products during rectal intercourse may increase the risk of infection with HSV-2 or other sexually transmitted pathogens.