SummaryBackgroundThe efficacy of on-demand HIV pre-exposure prophylaxis (PrEP) for men in sub-Saharan Africa has not been evaluated, and the on-demand PrEP dosing requirement for insertive sex remains unknown.MethodsHIV-negative males 13–24 years, requesting voluntary medical male circumcision (VMMC), were enrolled into an open-label randomised controlled trial (NCT03986970), and randomised 1:1:1:1:1:1:1:1:1 to control arm or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) over one or two days, and circumcised 5 or 21 h thereafter. The primary outcome was foreskin p24 concentrations followingex vivoHIV-1BaLchallenge. Secondary outcomes included peripheral blood mononuclear cell (PBMC) p24 concentration, and drug concentrations in foreskin tissue, PBMCs, plasma and foreskin CD4+/CD4-cells. In the control arm, post-exposure prophylaxis (PEP) activity of non-formulated tenofovir-emtricitabine (TFV-FTC) or TAF-FTC was assessed withex vivodosing 1, 24, 48 or 72 h post-HIV-1 challenge.Findings144 participants were analysed. PrEP with F/TDF or F/TAF preventedex vivoinfection of foreskins and PBMCs both 5 and 21 h after PrEP dosing. There was no difference between F/TDF and F/TAF (p24day15geometric mean ratio 1.06, 95% confidence interval: 0.65–1.74). Additionalex vivodosing did not further increase inhibition. In the control arm, PEPex vivodosing was effective up to 48 post-exposure diminishing thereafter, with TAF-FTC showing prolonged protection compared to TFV-FTC. Participants receiving F/TAF had higher TFV-DP concentrations in foreskin tissue and PBMCs compared with F/TDF, irrespective of dose and sampling interval; but F/TAF did not confer preferential TFV-DP distribution into foreskin HIV target cells. FTC-TP concentrations with both drug regimens were equivalent and ∼1 log higher than TFV-DP in foreskin.InterpretationA double dose of either F/TDF or F/TAF given once either 5 or 21 h beforeex vivoHIV-challenge provided protection across foreskin tissue. Further clinical evaluation of pre-coital PrEP for insertive sex is warranted.FundingEDCTP2,10.13039/100005564Gilead Sciences,10.13039/501100004359Vetenskapsrådet.