Biomarkers of semen exposure have historically played their most important role in forensics, i.e., determining whether ejaculation took place in the course of a crime. However, it is becoming increasingly recognized that biomarkers of semen exposure can be useful in the development of new vaginal methods of HIV/sexually transmitted infections (STI) prevention and contraception. This review is based on the presentations of Drs. Michael Coppola (ContraVac, Inc.), Maurizio Macaluso (Centers for Disease Control and Prevention), Andrezej Kulczycki (University of Alabama at Birmingham), Christine Mauck (CONRAD), Robin Maguire (Population Council), and the subsequent discussion led by Drs. Susan Ballagh (CONRAD) and Johan Melendez (Johns Hopkins University) during a session entitled "Biomarkers of semen exposure" held during the conference entitled "Biomarkers for evaluation of vaginal microbicides and contraceptives: Discovery and early validation," organized by CONRAD and the Alliance for Microbicide Development in November of 2006.