We assessed risk behaviour in a heterosexual cohort undergoing prescreening for the first Phase I/II HIV vaccine trials in Soweto. We developed a survey and collected self-reported data from HIV-negative potential volunteers. Of 488 participants, most were single and approximately half were from households with incomes below the poverty level. Males reported higher rates of heavy alcohol use (P < 0.001), marijuana use (P < 0.001) and other recreational drug use (P < 0.01). Males reported more sex partners than females in the previous six months (P < 0.001), as well as more casual/anonymous partners (P < 0.001) and one-night stands (P < 0.001). Multivariate analyses revealed substance use and male gender predicted higher risk behaviours, including <100% condom use with known/suspected HIV-positive partners, having casual/anonymous partners and having more than two partners. For this population, male volunteers may need increased risk-reduction counselling during Phase I/II trials and additional recruitment methods may be necessary to identify high-risk female volunteers for Phase III efficacy trials.