Background Twice-daily raltegravir with once-daily tenofovir-emtricitabine is an effective initial antiretroviral regimen for patients with HIV-1. On the basis of pharmacokinetic data suggesting efficacy of once-daily raltegravir and because adherence is often improved with once-daily dosing, we aimed to compare these dosing schedules. Methods In our international, double-blind, randomised, phase 3 non-inferiority study, we enrolled antiretroviral-naive patients with HIV RNA loads of more than 5000 copies per mL and no baseline resistance to tenofovir or emtricitabine at 83 centres worldwide. We randomly allocated patients (1:1) by use of a computer-generated sequence to receive raltegravir once daily (two 400 mg tablets taken together every 24 h), or twice daily (one 400 mg tablet every 12 h), both in combination with once-daily co-formulated tenofovir 300 mg plus emtricitabine 200 mg. The primary outcome was virological response at 48 weeks (viral RNA loads <50 copies per mL) in patients who received at least one dose of study drug, counting non-completers as failure. We assessed non-inferiority in terms of the proportion of patients in both treatment groups who achieved the primary outcome, with a non-inferiority margin of -10%. This study is registered with ClinicalTrials.gov, number NCT00745823. Findings From Oct 15, 2008, to Nov 2, 2009, we randomly allocated 775 patients, of whom 382 (99%) of 386 patients in the once-daily group and 388 (99%) of 389 in the twice-daily group received at least one dose of study drug. At baseline, 304 (39%) of 770 treated patients had viral loads of more than 100 000 copies per mL and 188 (24%) had CD4 cell counts of fewer than 200 cells per mu L. 318 (83%) of 382 patients in the once-daily group had virological response compared with 343 (89%) of 386 in the twice-daily group (difference -5.7%, 95% CI -10.7 to -0.83; p=0.044). Serious adverse events were reported in 26 (7%) of 382 once-daily recipients and 40 (10%) of 388 twice-daily recipients, and adverse events leading to discontinuation occurred in four (1%) patients in each group. Interpretation Despite high response rates with both regimens, once-daily raltegravir cannot be recommended in place of twice-daily dosing.