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In preparation for trials of new HIV prevention methods, willingness to participate (WTP) was assessed in Beira, Mozambique. A total of 1 019 women participating in an HIV incidence study, and 97 men participating in a separate WTP survey, were interviewed. When comparing the answers to questions that were identical in the two studies, WTP was higher among women than men for all prevention methods. Among women, WTP was highest for trials evaluating daily oral pre-exposure prophylaxis (PrEP; 84.4% reporting very likely to participate), followed by vaccination (77.8%), daily vaginal gel use (67.7%), coital vaginal gel use (67.1%) and monthly vaginal ring use (47.7%). Among men, WTP was highest for trials evaluating vaccination (57.6%), followed by daily vaginal gel use for female sexual partners (52.5%), daily oral PrEP (49.5%), coital vaginal gel use for female sexual partners (46.4%) and monthly vaginal ring use for female sexual partners (39.4%). Among men, the most important motivators for trial participation were social benefits, whereas personal risks (most notably receiving injections and/or blood draws) were deterrents; this was not assessed in women. Other important lessons learnt are that male circumcision and antiretroviral drugs were not generally recognised as ways to prevent HIV, that having to use hormonal contraception during trial participation will likely reduce WTP, and that evening clinics are not likely to be popular. The barriers reported in this and other studies may be challenging but are not impossible to overcome.